High throughput generation and affinity maturation of humanized antibody

ABSTRACT

Compositions, methods, and kits are provided for efficiently generating and screening humanized antibody with high affinity against a specific antigen. The library of humanized antibody is generated by mutagenizing a chimeric antibody template that combines human antibody framework and antigen binding sites of a non-human antibody. Alternatively, the library of humanized antibody is generated by grafting essential antigen-recognition segment(s) such as CDRs of the non-human antibody into the corresponding position(s) of each member of a human antibody library. This library of humanized antibody is then screened for high affinity binding toward a specific antigen in vivo in organism such as yeast or in vitro using techniques such as ribosome display or mRNA display. The overall process can be efficiently performed in a high throughput and automated manner, thus mimicking the natural process of antibody affinity maturation.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefit of U.S. ProvisionalApplication No. 60/403,296 filed Aug. 12, 2002, which is herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to compositions, methods and kits forgenerating libraries of humanized antibodies for the screening ofantibody with high affinity toward specific target antigens and reducedimmunogenicity in human, and, more particularly, for generation andaffinity maturation of the humanized antibody in a high throughput andautomated manner.

[0004] 2. Description of Related Art

[0005] Antibodies are a diverse class of molecules. Delves, P. J. (1997)“Antibody production: essential techniques”, New York, John Wiley &Sons, pp. 90-113. It is estimated that even in the absence of antigenstimulation a human makes at least 10¹⁵ different antibody molecules—itsPermian antibody repertoire. The antigen-binding sites of manyantibodies can cross-react with a variety of related but differentantigenic determinants, and the Permian repertoire is apparently largeenough to ensure that there will be an antigen-binding site to fitalmost any potential antigenic determinant, albeit with low affinity.

[0006] Structurally, antibodies or immunoglobulins (Igs) are composed ofone or more Y-shaped units. For example, immunoglobulin G (IgG) has amolecular weight of 150 kDa and consists of just one of these units.Typically, an antibody can be proteolytically cleaved by the proteinasepapain into two identical Fab (fragment antigen binding) fragments andone Fc (fragment crystallizable) fragment. Each Fab contains one bindingsite for antigen, and the Fc portion of the antibodies mediates otheraspects of the immune response.

[0007] A typical antibody contains four polypeptides-two identicalcopies of a heavy (H) chain and two copies of a light (L) chain, forminga general formula H₂L₂. Each L chain is attached to one H chain by adisulfide bond. The two H chains are also attached to each other bydisulfide bonds. Papain cleaves N-terminal to the disulfide bonds thathold the H chains together. Each of the resulting Fabs consists of anentire L chain plus the N-terminal half of an H chain; the Fc iscomposed of the C-terminal halves of two H chains. Pepsin cleaves atnumerous sites C-terminal to the inter-H disulfide bonds, resulting inthe formation of a divalent fragment [F(ab′)] and many small fragmentsof the Fc portion. IgG heavy chains contain one N-terminal variable(V_(H)) plus three C-terminal constant (C_(H)1, C_(H)2 and C_(H)3)regions. Light chains contain one N-terminal variable (V_(L)) and oneC-terminal constant (C_(L)) region each. The different variable andconstant regions of either heavy or light chains are of roughly equallength (about 110 amino residues per region). Fabs consist of one V_(L),V_(H), C_(H)1, and C_(L) region each. The V_(L) and V_(H) portionscontain hypervariable segments (complementarity-determining regions orCDR) that form the antibody combining site.

[0008] The V_(L) and V_(H) portions of a monoclonal antibody have alsobeen linked by a synthetic linker to form a single chain protein (scFv)which retains the same specificity and affinity for the antigen as themonoclonal antibody itself. Bird, R. E., et al. (1988) “Single-chainantigen-binding proteins” Science 242:423-426. A typical scFv is arecombinant polypeptide composed of a V_(L) tethered to a V_(H) by adesigned peptide, such as (Gly₄-Ser)₃, that links the carboxyl terminusof the V_(L) to the amino terminus of the V_(H) sequence. Theconstruction of the DNA sequence encoding a scFv can be achieved byusing a universal primer encoding the (Gly₄-Ser)₃ linker by polymerasechain reactions (PCR). Lake, D. F., et al. (1995) “Generation of diversesingle-chain proteins using a universal (Gly₄-Ser)₃ encodingoligonucleotide” Biotechniques 19:700-702.

[0009] The mammalian immune system has evolved unique genetic mechanismsthat enable it to generate an almost unlimited number of different lightand heavy chains in a remarkably economical way by joining separate genesegments together before they are transcribed. For each type of Igchain—κ light chains, λ light chains, and heavy chain—there is aseparate pool of gene segments from which a single peptide chain iseventually synthesized. Each pool is on a different chromosome andusually contains a large number of gene segments encoding the V regionof an Ig chain and a smaller number of gene segments encoding the Cregion. During B cell development a complete coding sequence for each ofthe two Ig chains to be synthesized is assembled by site-specificgenetic recombination, bringing together the entire coding sequences fora V region and the coding sequence for a C region. In addition, the Vregion of a light chain is encoded by a DNA sequence assembled from twogene segments—a V gene segment and short joining or J gene segment. TheV region of a heavy chain is encoded by a DNA sequence assembled fromthree gene segments—a V gene segment, a J gene segment and a diversityor D segment.

[0010] The large number of inherited V, J and D gene segments availablefor encoding Ig chains makes a substantial contribution on its own toantibody diversity, but the combinatorial joining of these segmentsgreatly increases this contribution. Further, imprecise joining of genesegments and somatic mutations introduced during the V-D-J segmentjoining at the pre-B cell stage greatly increases the diversity of the Vregions.

[0011] After immunization against an antigen, a mammal goes through aprocess known as affinity maturation to produce antibodies with higheraffinity toward the antigen. Such antigen-driven somatic hypermutationfine-tunes antibody responses to a given antigen, presumably due to theaccumulation of point mutations specifically in both heavy-andlight-chain V region coding sequences and a selected expansion ofhigh-affinity antibody-bearing B cell clones.

[0012] Great efforts have been made to mimic such a natural maturationof antibodies against various antigens, especially antigens associatedwith diseases such as autoimmune diseases, cancer, AIDS and asthma. Inparticular, phage display technology has been used extensively togenerate large libraries of antibody fragments by exploiting thecapability of bacteriophage to express and display biologicallyfunctional protein molecule on its surface. Combinatorial libraries ofantibodies have been generated in bacteriophage lambda expressionsystems which may be screened as bacteriophage plaques or as colonies oflysogens (Huse et al. (1989) Science 246: 1275; Caton and Koprowski(1990) Proc. Natl. Acad. Sci. (U.S.A.) 87: 6450; Mullinax et al (1990)Proc. Natl. Acad. Sci. (U.S.A.) 87: 8095; Persson et al. (1991) Proc.Natl. Acad. Sci. (U.S.A.) 88: 2432). Various embodiments ofbacteriophage antibody display libraries and lambda phage expressionlibraries have been described (Kang et al. (1991) Proc. Natl. Acad. Sci.(U.S.A.) 88: 4363; Clackson et al. (1991) Nature 352: 624; McCafferty etal. (1990) Nature 348: 552; Burton et al. (1991) Proc. Natl. Acad. Sci.(U.S.A.) 88: 10134; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133; Chang et al. (1991) J. Immunol. 147: 3610; Breitling et al. (1991)Gene 104: 147; Marks et al. (1991) J. Mol. Biol. 222: 581; Barbas et al.(1992) Proc. Natl. Acad. Sci. (U.S.A.) 89: 4457; Hawkins and Winter(1992) J. Immunol. 22: 867; Marks et al. (1992) Biotechnology 10: 779;Marks et al. (1992) J. Biol. Chem. 267: 16007; Lowman et al (1991)Biochemistry 30: 10832; Lerner et al. (1992) Science 258: 1313). Alsosee review by Rader, C. and Barbas, C. F. (1997) “Phage display ofcombinatorial antibody libraries” Curr. Opin. Biotechnol. 8:503-508.

[0013] Various scFv libraries displayed on bacteriophage coat proteinshave been described. Marks et al. (1992) Biotechnology 10: 779; Winter Gand Milstein C (1991) Nature 349: 293; Clackson et al. (1991) op.cit.;Marks et al. (1991) J. Mol. Biol. 222: 581; Chaudhary et al. (1990)Proc. Natl. Acad. Sci. (USA) 87: 1066; Chiswell et al. (1992) TIBTECH10: 80; and Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879.

[0014] Generally, a phage library is created by inserting a library of arandom oligonucleotide or a cDNA library encoding antibody fragment suchas V_(L) and V_(H) into gene 3 of M13 or fd phage. Each inserted gene isexpressed at the N-terminal of the gene 3 product, a minor coat proteinof the phage. As a result, peptide libraries that contain diversepeptides can be constructed. The phage library is then affinity screenedagainst immobilized target molecule of interest, such as an antigen, andspecifically bound phages are recovered and amplified by infection intoEscherichia coli host cells. Typically, the target molecule of interestsuch as a receptor (e.g., polypeptide, carbohydrate, glycoprotein,nucleic acid) is immobilized by covalent linkage to a chromatographyresin to enrich for reactive phage by affinity chromatography) and/orlabeled for screen plaques or colony lifts. This procedure is calledbiopanning. Finally, amplified phages can be sequenced for deduction ofthe specific peptide sequences. During the inherent nature of phagedisplay, the antibodies displayed on the surface of the phage may notadopt its native conformation under such in vitro selection conditionsas in a mammalian system. In addition, bacteria do not readily process,assemble, or express/secrete functional antibodies.

[0015] Transgenic animals such as mice have been used to generate fullyhuman antibodies by using the XENOMOUSE™ technology developed bycompanies such as Abgenix, Inc., Fremont, Calif. and Medarex, Inc.Annandale, N.J. Strains of mice are engineered by suppressing mouseantibody gene expression and functionally replacing it with humanantibody gene expression. This technology utilizes the natural power ofthe mouse immune system in surveillance and affinity maturation toproduce a broad repertoire of high affinity antibodies. However, thebreeding of such strains of transgenic mice and selection of highaffinity antibodies can take a long period of time. Further, the antigenagainst which the pool of the human antibody is selected has to berecognized by the mouse as a foreign antigen in order to mount immuneresponse; antibodies against a target antigen that does not haveimmunogenicity in a mouse may not be able selected by using thistechnology. In addition, there may be a regulatory issue regarding theuse of transgenic animals, such as transgenic goats (developed byGenzyme Transgenics, Framingham, Mass.) and chickens (developed byGeneworks, Inc., Ann Arbor, Mich.), to produce antibody, as well assafety issues concerning containment of transgenic animals infected withrecombinant viral vectors.

[0016] Antibodies and antibody fragments have also been produced intransgenic plants. Plants, such as corn plants (developed by IntegratedProtein Technologies, St. Louis, Mo.), are transformed with vectorscarrying antibody genes, which results in stable integration of theseforeign genes into the plant genome. In comparison, most microorganismstransformed with plasmids can lose the plasmids during a prolongedfermentation. Transgenenic plant may be used as a cheaper means toproduce antibody in large scales. However, due to the long growthcircles of plants screening for antibody with high binding affinitytoward a target antigen may not be efficient and feasible for highthroughput screening in plants.

[0017] Currently, the most efficient way of generation of non-humanantibody with high specificity and affinity is through using thehybridoma technology to produce monoclonal antibody against a specificantigen. The hybridoma technology invented by Milstein and Kohlerrevolutionized the industry of mass producing “custom-built” antibodiesin vitro. Basically, a hybridoma is generated by fusing rodent antibodyproducing cells with immortal tumor cells (myelomas) from the bonemarrow of mice. A hybridoma has the cancer cell's ability to reproducealmost indefinitely, as well as the immune cell's ability to secreteantibodies. The hybridomas producing antibodies of a determined antigenspecificity and required affinity were selected, expand in clonal sizeand mass-produce antibodies of a single type, i.e. monoclonalantibodies.

[0018] Compared to polyclonal antibodies produced from the serum ofanimals, monoclonal antibody generated in hybridoma is superior in termsof antigen selectivity, specificity and binding affinity. Owing to thesesuperior advantages associated with monoclonal antibodies, they havebeen hailed as “magic bullets” that could be used to specifically targetdiseased cells or tissues.

[0019] Although monoclonal antibodies (mAbs) generated from hybridomatechnology have proved to be immensely useful scientific research anddiagnostic tools, they have had a limited success in human therapy.Although murine antibodies had exquisite specificity for therapeutictargets, they did not always trigger the appropriate human effector'ssystems of complement and Fc receptors. More importantly, the majorlimitation in the clinical use of rodent monoclonal antibodies is anantiglobulin response during therapy. Miller et al. (1983) Blood62:988-995; and Schroff et al. (1985) Cancer Res. 54:879-885. Thepatient's immune system normally cuts short the therapeutic window, asmurine antibodies are recognized by a human anti-mouse antibody immuneresponse (HAMA). Similar to serum therapy where antisera used toneutralize pathogen in acute diseases and also prophylactically leads to“serum sickness”, the patient treated with rodent mAbs in multiple dosesinvariably raises an immune response to the mAbs, manifesting similarsymptoms to serum sickness and violent enough to endanger life. Thisresponse can occur within two weeks of the initiation of treatment andprecludes long-term therapy. Efforts have been made to raise human mAbsagainst therapeutic targets through immortalization of humanantibody-producing cells. The endeavors face various practical andethical problems, such as the difficulties with preparation of humanhybridomas that are unstable and secrete low levels of mAbs of the IgMclass with low affinity.

[0020] To produce therapeutic antibodies with high binding affinity,reduced immunogenicity (HAMA response), increased half-life in the humanbody and adequate recruitment of effectors functions (i.e. the abilityto summon help from the body's own natural defense), people in the arthave combined the techniques of monoclonal antibody production andrecombinant DNA technology to overcome the problem associated withrodent monoclonal antibodies. Besides direct generation of fully humanantibody as described above, another popular approach is to humanizerodent monoclonal antibody.

[0021] The technique of rodent antibody humanization takes advantage ofthe modular nature of antibody functions. It is based on the assumptionthat it's possible to convert a rodent, e.g., mouse, monoclonal antibodyinto one that has some human segments but still retains its originalantigen binding specificity. Such a chimeric antibody is humanized in asense that the mainframe of the antibody has human sequence whereas theantigen binding site have sequences derived from the counterparts of themouse monoclonal antibody.

[0022] Initially, the mouse Fc fragment was replaced with a humansequence because the mouse Fc functions poorly as an effector ofimmunological responses in humans; and it is also the most likelyfragment to elicit the production of human antibodies. To diminishimmunogenicity and to introduce human Fc effector capabilities, the DNAcoding sequences for the Fv regions of both the light and heavy chainsof a human immunoglobulin were substituted for the FvDNA sequences forthe light and heavy chains from a specific mouse monoclonal antibody.LoBuglio et al. (1989) Proc. Natl. Acad. Sci. USA 86:4220-4224. Thisreplacement of Fv coding regions can be accomplished by usingoligonucleotides and in vitro DNA replication or by using subclonalsegments. The DNA constructs for both chimeric chains were cloned intoan expression vector and transfected into cultured B lymphocytes fromwhich the chimeric antibody was collected.

[0023] Later the humanizing of mouse and rat monoclonal antibodies hasbeen taken one step further than the formation of chimeric moleculedescribed above by substituting into human antibodies on the CDRs of therodent antibodies, a process called “CDR grafting”. Queen et al. (1989)Proc. Natl. Acad. Sci. USA 86: 10029-10033. It was believed that such a“reshaped” human antibodies have antigen binding affinities similar tothose of the original rodent monoclonal antibodies and yet has a reducedimmunogenicity when used as a therapeutic agent in the clinic.Currently, CDR grafting is the most frequently used strategy for thehumanization of murine mAbs. In this approach the six CDR loopscomprising the antigen-binding site of the murine mAb are grafted intocorresponding human framework regions. However pure CDR-grafting oftenyields humanized antibodies with much lower affinity (Jones et al.(1986) Nature 321:522-525), in some instances as much as 10-fold ormore, especially when the antigen is a protein (Verhoeyen et al. (1988)Science 239:1534-1536). Such an antibody with reduced affinity isundesirable in that 1) more of the humanized antibody would have to beadministered into a patient at higher cost and greater risk of adverseeffects; 2) lower affinity antibody may have poorer biologicalfunctions, such as complement lysis, antibody-dependent cellularcytotoxicity, or virus neutralization. Riechmann et al. (1988) Nature332: 323-327.

[0024] To search for humanized antibody with higher affinity, Queen etal. have used computer modeling software to guide the humanization ofpromising murine antibodies. U.S. Pat. No. 5,693,762. The structure of aspecific antibody is predicted based on computer modeling and the fewkey amino acids in the framework are predicted to be necessary to retainthe shape, and thus the binding specificity, of the CDRs. These few keymurine amino acids are substituted into a human antibody framework alongwith the murine CDRs. As a result, the humanized antibody includes about90% human sequence. The humanized antibody designed by computer modelingis tested for antigen binding. Experimental results such as bindingaffinity are fed back to the computer modeling process to fine-tune thestructure of the humanized antibody. The redesigned antibody can then betested for improved biological functions. Such a reiterate fine tuningprocess can be labor intensive and unpredictable.

SUMMARY OF THE INVENTION

[0025] The present invention provides compositions, methods, and kitsfor efficiently generating and screening humanized antibody with highaffinity against a specific antigen. One feature of the presentinvention is that a library of humanized antibody is generated bymutagenizing a chimeric antibody template that combines human antibodyframework and antigen binding sites of a non-human antibody.

[0026] Alternatively, the library of humanized antibody is generated bygrafting essential antigen-recognition segment(s) of the non-humanantibody into the corresponding position(s) of each member of a humanantibody library. This library of humanized antibody is then screenedfor high affinity binding toward a specific antigen in vivo in organismsuch as yeast or in vitro using techniques such as ribosome display ormRNA display.

[0027] The specific antigen used in the screening can be the one againstwhich the non-human antibody is originally elicited, or an antigen withsimilar structural features or biological function. In addition, thelibrary of humanized antibody may be used in screening for high affinityantibody against an antigen that is structurally and/or functionallydifferent from the antigen against which the non-human antibody isoriginally elicited.

[0028] These selection processes can be performed to select antibodyhaving higher affinity in antigen binding but lower immunogenecity thanrodent monoclonal antibody. The overall process can be efficientlyperformed in a high throughput and automated manner, thus mimicking thenatural process of antibody affinity maturation.

BRIEF DESCRIPTION OF FIGURES

[0029]FIG. 1 illustrates the variable regions of the heavy chain andlight chain of a non-human antibody to be humanized. The CDR regionsbetween the framework of this antibody are labeled as CDR1, CDR2, andCDR3 sequentially from the N-terminus to the C-terminus.

[0030]FIG. 2 illustrates an example of a chimeric antibody havingnon-human CDRs 1-3 grafted into a human antibody framework.

[0031]FIG. 3 shows the DNA sequences of a consensus V_(H) (DP47) and aconsensus V_(L) (DPK22) of human antibody germine sequences.

[0032]FIG. 4A shows the DNA and amino acid sequences of V_(H) and V_(L)of a mouse monoclonal anti-interleukin-8 antibody (Murine IL-8 Ab).

[0033]FIG. 4B shows the amino acid sequences of V_(H) of human antibodyKabat Entry No: 037656 and V_(L) of human antibody Kabat Entry No:039682 which share high sequence homology to V_(H) and V_(L) of MurineIL-8 Ab in FIG. 4A, respectively.

[0034]FIG. 5A shows alignment of V_(H) of murine IL-8 Ab shown in FIG.4A and V_(H) of human antibody Kabat Entry No: 037656 shown in FIG. 4B.

[0035]FIG. 5B shows alignment of V_(L) of murine IL-8 Ab shown in FIG.4A and V_(L) of human antibody Kabat Entry No: 039682 shown in FIG. 4B.

[0036]FIG. 6 illustrates an embodiment of the method for generating,expressing, and screening in yeast a library of humanized antibody intowhich the CDRs of non-human antibody are grafted.

[0037]FIG. 7 illustrates an embodiment of the method for generating,expressing, and screening in yeast a library of humanized antibody intowhich CDR3 of non-human antibody is grafted.

[0038]FIG. 8 illustrates an embodiment of the method for generating,expressing, and screening in yeast a library of fully human antibody,which is directed by V_(H) of a non-human antibody.

[0039]FIG. 9 illustrates an embodiment of the method for selectinghumanized single-chain antibody (scFv) against a target protein in atwo-hybrid system where the expression vectors carrying the AD and BDdomains are co-transformed or sequentially transformed into yeast.

[0040]FIG. 10 illustrates an embodiment of the method for selectinghumanized single-chain antibody (scFv) against a target protein in atwo-hybrid system where the expression vectors carrying the AD and BDdomains are introduced into diploid yeast cells via mating between twohaploid yeast strains of opposite mating types.

[0041]FIG. 11 illustrates an embodiment of the method for selectinghumanized antibody against a target antigen through ribosome display.

[0042]FIG. 12 illustrates an embodiment of the method for selectinghumanized antibody against a target antigen through mRNA display.

[0043]FIG. 13 illustrates an embodiment of the method used formutagenesis and further screening of the clones selected from a primaryscreening of the humanized antibody in yeast.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention provides novel methods for efficientlygenerating and screening humanized antibody with high affinity against aspecific antigen. Compared to approaches that use stepwise tailoring anddesigning of individual humanized antibody in silicon (i.e. computermodeling), the humanization process according to the present inventionis performed in vitro or in vivo and screened directly against thetarget antigen. Therefore, the present approach is more robust and moredirectly mimics the natural process of antibody affinity maturation invertebrates. By using the methods of the present invention, non-humanantibody can be humanized not only without loss in antigen-bindingaffinity but also with improved affinity and other biological functions.The whole process of antibody humanization and affinity maturation canbe performed in a high throughput manner.

[0045] In one aspect of the present invention, a method is provided forhumanizing a non-human antibody by mutagenesis. The method comprises:constructing a chimeric antibody sequence by combining a human antibodyframework sequence with one or more non-human antibody segments that areessential for affinity binding to a target antigen against which thenon-human antibody is elicited; mutagenizing the chimeric antibodysequence to produce a library of humanized antibody sequences.

[0046] In another aspect of the present invention, a method is providedfor humanizing a non-human antibody by grafting non-human antibodysegments into a library of human antibody sequences. The methodcomprises: grafting one or more non-human antibody segments into alibrary of human antibody framework sequences to produce a library ofhumanized antibody sequences. The non-human antibody segments graftedare essential for affinity binding to a target antigen against which thenon-human antibody is elicited.

[0047] The library of humanized antibody sequences are expressed invitro or in vivo to produce a library of humanized antibodies which canbe screened for high affinity bind to a target antigen. In oneembodiment, the library of humanized antibody sequences is expressed invivo and screened against the target antigen in yeast, preferably in ayeast two-hybrid system. In another embodiment, the library of humanizedantibody sequences is expressed and screened in vitro against the targetantigen, preferably by ribosome display.

[0048] It should be noted that the specific antigen used in thescreening can be the one against which the non-human antibody isoriginally elicited, or an antigen with similar structural features orbiological function.

[0049] These selection processes can be performed to select antibodyhaving higher affinity in antigen binding but lower immunogenecity thanrodent monoclonal antibody. In contrast to the approach using computermodeling of individually humanized antibody and subsequent experimentalscreening, the overall process of the present invention can beefficiently performed in a high throughput and automated manner, thusmimicking the natural process of antibody affinity maturation.

[0050] The present invention provides methods for producing andscreening humanized antibody with high affinity and specificity. Themethods are efficient, comprehensive and complementary.

[0051] First, the method of producing and screening an antibody libraryin yeast is an efficient and economical way to screen for humanizedantibodies in a much shorter period of time. In addition, production ofthe library of humanized antibody sequence can be carried out in a highthroughput manner in yeast by exploiting the intrinsic genetic propertyof yeast—homologous recombination at an extremely high level ofefficiency. This process will be described in details in Section 2below.

[0052] The fast proliferation rate of yeast cells and ease of handlingmakes a process of “molecular evolution” dramatically shorter than thenatural process of antibody affinity maturation in a mammal. Therefore,humanized antibody repertoire can be produced and screened directly inyeast cells at a much lower cost and higher efficiency than priorprocesses such as the painstaking, stepwise “humanization” of monoclonalmurine antibodies isolated by using the conventional hybridomatechnology (a “protein redesign”) or the XENOMOUSE™ technology.

[0053] According to the “protein redesign” approach, murine monoclonalantibodies of desired antigen specificity are modified or “humanized” invitro in an attempt to reshape the murine antibody to resemble moreclosely its human counterpart while retaining the originalantigen-binding specificity. Riechmann et al. (1988) Nature 332:323-327.This humanization demands extensive, systematic and reiterate computerengineering and experimental validation of the murine antibody, whichcould take months, if not years. In addition, this approach can bear therisk of empirical guessing or wrong prediction based on sequencecomparison and structural modeling.

[0054] In comparison, by using the method of the present invention,humanized antibodies with perhaps even higher affinity to a specifiedantigen than the original non-human antibody can be screened andisolated directly from yeast cells without going through reiteratesite-by-site computer engineering and experimental validation. Thelibrary of humanized antibody can diversified by site-directed or randommutagenesis and directly screened against the target antigen in vivo inyeast or via ribosome display in vitro. The selected humanized antibodycan be further mutagenized and screened again for higher affinity binderto the same target antigen. This reiterate process mimics the naturalprocess of antibody maturation in vertebrates.

[0055] Further, by using the method of the present invention, manyrequisite steps in the traditional construction of cDNA libraries can beeliminated. For example, the time-consuming and labor-intensive steps ofligation and recloning of cDNA libraries into expression vectors can beeliminated by direct recombination or “gap-filling” in yeast throughgeneral homologous recombination and/or site-specific recombination.Throughout the whole process of humanized antibody library construction,the DNA fragments encoding antibody heavy chain and light chain aredirectly incorporated into a linearized yeast expression vector viahomologous recombination without the recourse to extensive recloning.

[0056] Moreover, the library of humanized antibody can also be screenedagainst an array of antigens to identify those which bind to a specificantigen in the array with the highest affinity.

[0057] In addition, by using the method of present inventions, multiplehumanized antibody may be selected against the same target antigen. Inclinical therapeutic applications, if the one of these antibodieselicits an anti-idiotypic response in the patient, another one from thesame group of antibodies can be used to substitute the idiotypic one,thus allowing the therapy to continue without ablating the therapeuticefficacy.

[0058] Second, the methods are more comprehensive than the XENOMOUSE™technology. The XENOMOUSE™ technology has been used to generate fullyhuman antibodies with high affinity by creating strains of transgenicmice that produce human antibodies while suppressing the endogenousmurine Ig heavy- and light-chain loci. However, the breeding of suchstrains of transgenic mice and selection of high affinity antibodies cantake a long period of time. The antigen against which the pool of thehuman antibody is selected has to be recognized by the mouse as aforeign antigen in order to mount immune response; and antibodiesagainst a target antigen that does not have immunogenicity in a mousemay not be able to be selected by using this technology.

[0059] In contrast, by using the method of the present invention,libraries of humanized antibody can not only be generated in yeast cellsmore efficiently and economically, but also be screened againstvirtually any protein or peptide target regardless of itsimmunogenicity. According to the present invention, any protein/peptidetarget can be expressed as a fusion protein with a DNA-binding domain(or an activation domain) of a transcription activator and selectedagainst the library of antibody in a yeast-2-hybrid system.

[0060] Third, the methods provided by the present invention arecomplementary. On one hand, a yeast two-hybrid system can be used toscreen for high affinity humanized antibody against any protein antigenexpressed intracellularly. On the other hand, a ribosome display methodcan be used to display the library of humanized antibody on the surfaceof ribosomes and screened for virtually any ligand. Since the ribosomedisplay is performed by in vitro translation of mRNA encoding thelibrary of humanized antibody in a cell lysate, the library of humanizedantibody bound to the ribosomes can be screened against any ligandimmobilized on a substrate. The immobilized ligand can be a smallmolecule, a peptide, a protein, and a nucleic acid.

[0061] The preferred embodiments of the methods for generation andaffinity maturation of humanized antibody are described as follows.

[0062] 1. Generation of a Library of Humanized Antibody

[0063] The present invention provides methods for generating a libraryof humanized antibody that can be used for screening for antibody withhigh affinity toward a specific antigen. The humanized antibodies in thelibrary contain a human framework and essential antigen bindingsegment(s) derived from a non-human antibody, such as a mouse or ratantibody. The following are examples of how to generate such a libraryof humanized antibody

[0064] 1) Construction of a library of humanized antibody by creating achimeric antibody by grafting essential antigen recognition segments ofa non-human antibody into a single human antibody framework, andmutagenizing the chimeric antibody

[0065] In this embodiment, the library of humanized antibody sequencesis constructed by grafting sequences encoding essential antigen-bindsegments (e.g., CDRs) of a non-human antibody (e.g., a mouse monoclonalantibody) into the sequence encoding a single human antibody framework.Through this grafting process, a chimeric antibody sequence is createdto encode a chimeric antibody including both human and non-humanantibody sequences. The chimeric antibody sequence is mutagenized toproduce a library of humanized antibody sequences.

[0066]FIG. 1 illustrates the variable regions of the heavy chain andlight chain of a non-human antibody. As illustrated by FIG. 1, thesegments that most likely determine the antigen-binding affinity of thenon-human antibody are CDR regions, including CDR 1, CDR2, and CDR3located in the variable regions of the heavy chain and light chain. Therest of the sequences of the variable regions of the heavy chain andlight chain constitute the framework sequences of the antibody.

[0067]FIG. 2 illustrates the variable regions of the heavy chain andlight chain of a chimeric antibody. The sequences encoding the CDRregions of a non-human antibody (as shown in FIG. 1) are grafted intothe variables regions of a human antibody by replacing the human CDRs intheir corresponding positions. As a result, a chimeric antibody sequenceis created, including both human and non-human antibody sequences.

[0068] In this chimeric antibody the human antibody framework sequenceserves as a framework to accommodate the non-human CDRs and providesstructural support for global folding of the antibody structure. Thehuman framework sequence may be chosen based on various criteria.

[0069] For example, a fixed human antibody framework sequence may beused to provide the structural support for the chimeric antibody. Inthis case, a single vector containing the chosen human antibodyframework can be created to accept all non-human CDRs, generatinghumanized antibodies with similar expression and performance.

[0070] The fixed human antibody framework sequence may be derived fromnatural human antibodies, such as framework “NEW” (Saul F A et al.“Preliminary refinement and structural analysis of the Fab fragment fromhuman immunoglobulin NEW at 2.0 A resolution” J Biol Chem (1978) 253(2):585-597; and Riechmann et al. “Reshaping human antibodies for therapy.”Nature (1988) 332: 323-327) for the heavy chain and framework “REI” (Eppet al. “Crystal and molecular structure of a dimer composed of thevariable portions of the Bence-Jones protein REI.” Eur J Biochem. (1974)45(2):513-524; and Riechmann et al. “Reshaping human antibodies fortherapy.” Nature (1988) 332: 323-327) for the light chain. Althoughthese human antibodies are well characterized, using the frameworks fromparticular human antibodies for humanization may run a risk of somaticmutation that creates immunogenic epitopes.

[0071] In a preferred embodiment, the frameworks from human antibodyconsensus sequences where idiosyncratic somatic mutations have been“evened out” are used to provide the human antibody frameworks of thepresent invention. Kabat et al. “Sequences of Proteins of ImmunologicalInterest” Fifth Edition. (1991) NIH Publication No. 91-3242; andKolbinger F, Saldanha J, Hardman N and Bendig M “Humanization of a mouseanti-human IgE antibody: a potential therapeutic for IgE-mediatedallergies” Prot. Engng. (1993) 6: 971-980.

[0072] In another preferred embodiment, the human framework sequence isderived from consensus human germline sequences. Human antibodies areassembled from 51 different functional V_(H) germ line genes and 70different functional V_(L) segments (40 Vκ and 30 Vλ). However, oneV_(H) (DP47, its DNA SEQ ID NO: 1) and one Vκ (DPK22, its DNA SEQ ID NO:2) dominate the functional repertoire (Kirkham, P. M. et al. (1992) EMBOJ. 11:603-609). FIG. 3 shows the DNA sequences of DP47 and DPK22.

[0073] These two germ line gene segments are used as frameworks for CDRgrafting. The gene sequences are examined for all possible restrictionendonuclease sites, which could be introduced without changing thecorresponding amino acid sequences. Cleavage sites are chosen that arelocated close to the CDR and framework borders and are unique. Theresulting gene fragments are assembled from overlapping oligonucleotideson alternating strands by overlap-extension PCR. By cloning thesesynthesized gene fragments into appropriate vectors, two modularcassettes are generated into which any either heavy chain or light chainCDRs can be easily inserted. The donor CDRs will be individuallyamplified by PCR using primers that introduce restriction sitescompatible to those in the framework cassettes. The CDRs will then begrafted into the frameworks by restriction digestion and ligation.

[0074] For example, mouse monoclonal antibody against interleukin-8(Murine IL-8 Ab, ATCC No: HB-9647, Yoshimura et al. (1989) “Three formsof monocyte-derived neutrophil chemotactic factor (MDNCF) distinguishedby different lengths of the amino-terminal sequence” Mol. Immunol. 26:87-93; and Sylvester et al. (1990) “Secretion of neutrophilattractant/activation protein by lipopolysaccharide-stimulated lungmacrophages determined by both enzyme-linked immunosorbent assay andN-terminal sequence analysis” Am. Rev. Respir. Dis. 141: 683-688.) maybe humanized by grafting its CDR regions into a human antibody frameworksuch as DP47 for heavy chain and DPK22 for light chain, respectively.FIG. 4A shows the DNA and amino acid sequences of V_(H) and V_(L) ofMurine IL-8 Ab (murine V_(H): DNA [SEQ ID NO: 4] and protein [SEQ ID NO:5]; and murine V_(L): DNA [SEQ ID NO: 6] and protein [SEQ ID NO: 7]. Theresulting chimeric antibody may be mutagenized throughout the variableregion to produce a library of humanized antibodies which are thenscreened for antibodies with high affinity toward a specific target,such as IL-8.

[0075] Alternatively, the CDRs of the non-human antibody may be graftedinto a human framework through a homology match. In another word, aminoacid sequences of human antibody framework sequences are searched forbest homology with that of the non-human antibody to be humanized. Thehomology may be searched within an appropriate database of either humanantibodies or human germline sequences. Ideally, the human antibodychosen should share the highest percentage identity with the non-humanantibody in the length of the CDRs and the canonical residue. Once theframework sequences of the human antibody are chosen, the humanizedV_(H) and V_(L) genes are assembled from overlapping oligonucleotides byoverlap-extension PCR.

[0076] For example, the amino acid sequence of the Murine IL-8 Abdescribed above may be aligned with human antibody frameworks within ahuman antibody database, such as the Kabat database of human antibody.FIG. 4B shows the amino acid sequences of V_(H) of a human antibodyagainst CD19 (Kabat Entry No: 037656, Bejcek et al. (1995) “Developmentand characterization of three recombinant single chain antibodyfragments (scFvs) directed against the CD19 antigen” Cancer Res.55:2346-51) within the Kabat database which shares a high percentageidentity (85.5%) with the Murine IL-8 Ab in the framework regions. FIG.4B also shows the amino acid sequences of V_(L) of a human antibodyagainst the dominant epitope of the group A Streptococcal carbohudrate,N-acetyl-beta-D-glucosamine, (Kabat Entry No: 039682, Adderson et al.(1998) “Molecular analysis of polyreactive monoclonal antibodies fromrheumatic carditis: human anti-N-acetylglucosamine/anti-myosin antibodyV region genes” J Immunol. 161:2020-31) within the Kabat database whichshares a high percentage identity (80.2%) with the Murine IL-8 Ab in theframework regions.

[0077]FIGS. 5A and 5B show amino acid sequence alignments of V_(H) andV_(L) of the Murine IL-8 Ab (HB-9647) with V_(H) of human antibody KabatEntry No: 037656 and V_(L) of human antibody Kabat Entry No: 039682 inthe framework region, respectively. Amino acid residues that are nothomologous to those of the Murine IL-8 Ab in the framework regions arein bold.

[0078] As shown in FIG. 5A, CDR regions designated by Kabat in V_(H)region are framed in boxes (CDR1, aa 31-35B; CDR2, a 50-65; and CDR3, aa95-102) while those designated by Chothia are highlighted in gray areas(CDR1, aa 26-32; CDR2, aa 52-56; and CDR3, aa 95-102).

[0079] As shown in FIG. 5B, CDR regions designated by Kabat in V_(L)region are framed in boxes (CDR1, aa 24-34; CDR2, aa 50-56; and CDR3, aa89-97) while those designated by Chothia are highlighted in gray areas(CDR1, aa 24-34; CDR2, aa 50-56; and CDR3, aa 89-96).

[0080] As shown in FIG. 5A, V_(H) of human antibody Kabat Entry No:037656 shares a very high sequence homology (85.5%) with that of theMurine IL-8 Ab in the framework region of V_(H). As shown in FIG. 5B,V_(L) of human antibody Kabat Entry No: 039682 shares a very highsequence homology (80.2%) with that of the Murine IL-8 Ab in theframework region of V_(L). Thus, the frameworks of V_(H) of humanantibody Kabat Entry No: 037656 and V_(L) of human antibody Kabat EntryNo: 039682 can serve as the frameworks to accommodate the CDR regions ofthe Murine IL-8 Ab in V_(H) and V_(L) regions, respectively. Preferably,the CDR sequences that are selected to be grafted into the humanframework are the maximized CDR sequences including both Kabat andChothia CDRs. For the Murine IL-8 Ab, the CDRs to be grafted into theframeworks of V_(H) of human antibody Kabat Entry No: 037656 and V_(L)of human antibody Kabat Entry No: 039682 are as follows:

[0081] For V_(H), CDR1, aa 26-35B; CDR2, aa 50-65; and CDR3, aa 95-102.

[0082] For V_(L), CDR1, aa 24-34; CDR2, aa 50-56; and CDR3, aa 89-97.

[0083] The resulting chimeric antibody may be mutagenized throughout thevariable region to produce a library of humanized antibodies which arethen screened for antibodies with high affinity toward a specifictarget, such as IL-8.

[0084] The humanized V_(H) and V_(L) genes that combines the humanframework sequence and the non-human CDRs of the heavy chain and lightchain, respectively, may cloned into an expression vector or into twoexpression vectors separately. In this design, the V_(H) and V_(L) genescan be expressed to form a double chain chimeric antibody (dcFv).

[0085] Alternatively, the humanized V_(H) and V_(L) genes may beassembled by PCR to form a single chain chimeric antibody (scFv).Specifically, the V_(H) and V_(L) gene fragments generated above areassembled into a single fragment by PCR which adds a linker betweenV_(H) and V_(L). A typical linker region for a single chain antibody is4 tandem repeats of (GlyGlyGlyGlySer) [SEQ ID NO: 3].

[0086] During the PCR assembly, mutagenesis is introduced into thesingle chain chimeric antibody sequence. For example, error-prone PCRcan be used in this process to incorporate random mutations throughoutthe reading frames in both the heavy chain and light chain of thechimeric antibody sequence. As a result, a library of humanized antibodysequences is constructed.

[0087]FIG. 6 illustrates an example of the method for constructing alibrary of humanized antibody sequences contained in a yeast expressionvector. As illustrated in FIG. 6, the framework sequences of a humanlight chain and a heavy chain are separately contained in a cloningvector (e.g, pUC19). CDR sequences from a non-human antibody are graftedinto the framework sequence at the individual, unique restriction sitesin the corresponding positions of the human CDR regions. These chimericheavy chain and light chain sequences contained in the cloning vectorsare assembled by PCR in the presence of a linker sequence to form achimeric scFv fragment. During the PCR assembly process randommutagenesis is also performed to introduce mutations into the chimericscFv. As a result, a library of humanized antibody sequences isgenerated.

[0088] As illustrated in FIG. 6, the PCR primers are designed to includesequences flanking the chimeric scFv that can facilitate subsequenthomologous recombination of the scFv into a yeast expression vector. Thelibrary of humanized antibody sequences generated by PCR assembly isthen cloned into a yeast expression vector, such as a yeast two-hybridvector containing an activation domain (e.g, pACT2, Clontech, Palo Alto,Calif.). The two-hybrid vector is linearized with a single restrictionenzyme in the multiple cloning site (MCS). The library of humanizedantibody may then be co-transformed with the linearized vector into acompetent yeast strain. The successful homologous recombination shouldgenerate a library of mutagenzied scFvs fused with the activationdomain. High affinity mutants can be isolated from this library in ayeast two-hybrid screening. The process of yeast homologousrecombination and two-hybrid screening is described in more details inSection 2.

[0089] 2) Construction of a library of yeast expression vectorscontaining humanized antibody sequences by grafting essential antigenrecognition segments of a non-human antibody into a library of humanantibody sequences.

[0090] In this embodiment, the library of humanized antibody sequencesis constructed by grafting sequences encoding essential antigenrecognition segments (e.g., CDRs) of a non-human antibody (e.g., a mousemonoclonal antibody) into the framework sequences of a library of humanantibody sequences. Through this grafting process, a library of chimericantibody sequences is created to encode a library of chimeric antibodiesincluding both human and non-human antibody sequences. Such a library ofchimeric antibody sequences is cloned into a yeast expression vector togenerate a library of yeast expression vectors containing humanizedantibody sequences.

[0091] This humanization strategy involves two selection steps for thesequential humanization of the light chain and the Fd fragment of theheavy chain. Throughout these selections the only preserved sequences inthe variable domains are two of the six CDRs, LCDR3 of V_(L) and HCDR3of V_(H). FIG. 7 illustrates an example of the method of constructing alibrary of humanized antibody sequences containing only CDR3 regions ofthe non-human antibody.

[0092] In the first step, the light chain of the non-human antibody ishumanized by incorporating the non-human LCDR3 sequence into a libraryof human antibody V_(L) sequences. Degenerate PCR primers are used toamplify the fragment encoding framework 1 (FR1) through framework 3(FR3) from a human antibody library. By overlap-extension PCR, thisfragment is then fused with a PCR fragment encoding the LCDR3 of thenon-human antibody coupled to FR4 of human Vκ and the human Cκ domain.The FR4 of the human antibody can be chosen based on homology to thenon-human FR4; and changes in this region should have little effect onthe affinity. This process generates a library of human light chain thatcontains the LCDR3 of the non-human antibody. These PCR fragments can becloned into a yeast two-hybrid vector containing the activation domain(AD).

[0093] A chimeric heavy chain Fd fragment can be generated by fusing thenon-human V_(H) with human C_(H)1 and cloned into the same two-hybridvector. A zipper or bundle domain (described in detail below) may befused to V_(H) and V_(L) of the chimeric antibody to facilitate assemblyof these two fragments in yeast. By using the yeast two-hybrid screeningmethod (described in detail in Section 2), chimeric Fab with highaffinity toward the target antigen can be selected.

[0094] In the second step, the heavy chain of the non-human antibody ishumanized by incorporating the non-human HCDR3 sequence into a libraryof human antibody V_(H) sequences (FIG. 7). Following a similarprocedure to that in step 1, a library of human Fd fragment that containthe HCDR3 of the non-human antibody can constructed. These fragments arethen cloned into the yeast two-hybrid vector containing the humanizedlight chain selected from step 1. A second round of screening will leadto the selection of humanized Fab with high affinity toward to thetarget antigen.

[0095] In this preferred embodiment, a yeast two-hybrid vectorcontaining an activation domain (e.g., pACT2, Clontech, Palo Alto,Calif.) is modified to express Fab fragment, each composed of a chimericheavy chain and a chimeric light chain from the libraries describedabove. In the Fab fragment, one or more human constant regions (Cκ ofthe light chain and CH1 of the heavy chain) are included to stabilizethe Fab of the selection steps through intermolecular interactionsbetween the two matching human constant regions.

[0096] Alternatively, V_(H) and V_(L) can be expressed as fusionproteins with a zipper domain or a bundle domain to facilitate assemblyof V_(H) and V_(L) to form a stable Fab.

[0097] A zipper domain is a protein or peptide structural motif thatinteracts with each other through non-covalent interactions such ascoiled-coil interactions and brings other proteins fused with the zipperdomains into close proximity. Examples of zipper domains include, butare not limited to, leucine zippers (or helix-loop-helix, also calledbHLHzip motif) formed between the nuclear oncoproteins Fos and Jun(Kouzarides and Tiff (1989) “Behind the Fos and Jun leucine zipper”Cancer Cells 1: 71-76); leucine zippers formed betweenproto-oncoproteins Myc and Max (Luscher and Larsson (1999) “The basicregion/helix-loop-helix/leucine zipper domain of Myc proto-oncoproteins:function and regulation” Oncogene 18:2955-2966); zipper motifs fromadhesion proteins such as N-terminal domain of neural cadherin (Weis(1995) “Cadherin structure: a revealing zipper” 3:425-427); zipper-likestructural motifs from collagen triple helices or cartilage oligomericmatrix proteins (Engel and Prockop “The zipper-like folding of collagentriple helices and the effects of mutations that disrupt the zipper”Annu. Rev. Biophys. Biophys. Chem. 20:137-152; and Terskikh et al.(1997) “Peptabody”: a new type of high avidity binding protein” Proc.Natl. Acad. Sci. USA 94:1663-1668).

[0098] The zipper domain may be fused to the N- or C-terminus of thehumanized antibody V_(H) or V_(L), preferably at the C-terminus of thesubunits. For example, the leucine zipper domain derived from theoncoprotein Jun can be expressed as a fusion protein with V_(H) whereasthe leucine zipper domain derived from the oncoprotein Fos can beexpressed as another fusion protein with V_(L). Since the Jun and Fosleucine zipper domains can bind to each other with high affinity, theantibody heavy chain and light chain fused with Jun and Fos zipper,respectively, can be brought into close proximity and form a heterodimerupon binding between these two zipper domains.

[0099] It is believed that by adding a zipper domain near the termini ofthe subunits, the intermolecular interactions between the two subunitsshould be enhanced through non-covalent interactions (e.g. hydrophobicinteractions), thus further stabilizing the assembly of Fab formed bythe humanized V_(H) and V_(L). Moreover, fusing a zipper domain derivedfrom nuclear protein such as Jun and Fos to V_(H) and V_(L) mayfacilitate efficient transportation of V_(H) and V_(L) to the nucleuswhere the Fab formed between the V_(H) and V_(L) performs desiredfunctions such as transcriptional activation of a reporter gene.

[0100] As used herein, a “bundle domain” refers to a protein or peptidestructural motif that can interact with itself to form a homo-polymersuch as a homopentalmer. The bundle domains bring the protein complextogether by polymerization through non-covalent interactions such ascoiled-coil interactions. It is believed that polymerization of theV_(H) and V_(L) should enhance the avidity of the Fab to their bindingtarget through multivalent binding.

[0101] For example, the coiled-coil assembly domain of the cartilageoligomeric matrix protein (COMP) may serve as a bundle domain. TheN-terminal fragment of rat COMP comprises residue 20-83. This fragmentcan form pentamers simillar to the assembly domain of the nativeprotein. The fragment adopts a predominantly alpha-helical structure.Efimov et al. (1994) “The thrombospondin-like chains of cartilageoligomeric matrix protein are assembled by a five-stranded alpha-helicalbundle between residues 20 and 83” FEBS Lett. 341:54-58.

[0102] The coiled-coil domain of the nudE gene of the filamentous fungusAspergillu nidulans or the gene encoding the nuclear distributionprotein RO11 of Neurospora crassa may also serve a bundle domain. Theproduct of the nudE gene, NUDE, is a homologue of the RO11 protein. TheN-terminal coiled-coil domain of the NUDE protein is highly conserved;and a similar coiled-coil domain is present in several putative humanproteins and in the mitotic phosphoprotein 43 (MP43) of X laevis. Efimovand Morris (2000) “The LIS1-related NUDF protein of Aspergillu nidulansinteracts with the coiled-coil domain of the NUDE/RO11 protein” J. CellBiol. 150:681-688.

[0103] In addition, the coiled-coil segments or fribritin encoded bybacteriophage T4 may also serve as a bundle domain. The bacteriophage T4late gene wac (Whisker's antigen control) encodes a fibrous proteinwhich forms a collar/whiskers complex. Analysis of the 486 amino acidsequence of fibritin reveals three structural components: a 408 aminoacid region that contains 12 putative coiled-coil segments with acanonical heptad (a-b-c-d-e-f-g)n substructure where the “a” and “d”positions are preferentially occupied by apolar residues, and the N andC-terminal domains (47 and 29 amino acid residues, respectively). Thealpha-helical segments are separated by short “linker” regions, variablein length, that have a high proportion of glycine and proline residues.Co-assembly of full-length fibritin and the N-terminal deletion mutant,as well as analytical centrifugation, indicates that the protein is aparallel triple-standard alpha-helical coiled-coil. The last 18C-terminal residues of fibritin are required for correct trimerisationof gpwac monomers in vivo. Efimov et al. (1994) “Fibritin encoded bybacteriophage T4 gene wac has a parallel triple-stranded alpha-helicalcoiled-coiled structure” J. Mol. Biol. 242:470-486.

[0104] 3) Construction of a library of fully human antibody sequencesdirected by essential antigen recognition segment(s) of a non-humanantibody

[0105] In this embodiment, a library of fully human antibody sequencesis constructed by a directed selection from two separate pools of fullyhuman antibody light chain and heavy chain sequences. The selection isdirected toward a chimeric antibody heavy chain comprising essentialantigen recognition segments (e.g., V_(H) or CDRs of the heavy chain) ofa non-human antibody and a human framework sequence such as a constantregion of a human antibody. The light chains from human antibody genepool and the chimeric heavy chain are expressed and assembled in vivo toform a library of chimeric Fab. This library of double chain Fabcontaining the chimeric light chain is selected against the originalantigen against which the non-human antibody is elicited. The fullyhuman light chain(s) of the chimeric Fab(s) selected in this process isthen matched with a pool of fully human antibody heavy chain sequencesto form a library of fully human antibody sequences. This library isscreened against the original antigen again to select for those fullyhuman antibodies with high affinity toward the original antigen. As aresult, the selected antibody is not only fully human but also may havepotentially higher affinity toward the antigen than the originalnon-human antibody. This fully human antibody should have the advantageof being less immunogenic than a chimeric antibody which includespartially human and partially non-human sequences.

[0106] The cDNA gene pool for the heavy chain or light chain of fullyhuman antibody may generated by using the methods known in the art.Sambrook, J., et al. (1989) Molecular Cloning: a laboratory manual. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; and Ausubel, F. M.et al. (1995) Current Protocols in Molecular Biology” John Wiley & Sons,NY.

[0107] Total RNA may be isolated from sources such as the white cells(mainly B cells) contained in peripheral blood supplied by unimmunizedhumans, or from human fetal spleen and lymph nodes. First strand cDNAsynthesis may be synthesized performed by using methods known in theart, such as those described by Marks et al. Marks et al. (1991) Eur. J.Immunol. 21:985-991.

[0108]FIG. 8 illustrates an example of the method for constructing alibrary of fully human antibody sequences. The library construction isdirected by essential antigen recognition segments (e.g., V_(H)) of anon-human antibody. According to this method, the process of humanizinga non-human antibody involves two steps of sequential humanization andlibrary screening.

[0109] In the first step, the light chain of a non-human antibody ishumanized. A library of chimeric antibody sequences is constructed witha bias toward a non-human V_(H). As illustrated in FIG. 8, V_(H) of thenon-human antibody to be humanized is linked to the human constantdomain 1 of the heavy chain, CH1, to form a chimeric antibody heavychain. A library of human Vλ and Vκ (or CDRs) is linked to the humanconstant domain of the light chain, CL, in a yeast two-hybrid vector.This library of human antibody light chain sequences can be expressed togenerate a library of chimeric Fabs by assembling with the chimericheavy chain expressed from a separate vector in yeast. Alternatively,the library of human antibody light chain and the chimeric heavy chainmay be expressed from separate expression cassettes in the same yeastvector. The assembly of these two chains may be facilitated by using“zipper” domains such as the Jun/Fos pair that are fused to the terminusof the heavy chain and light chain, respectively. The assembly ofantibody fragments by zipper domains is described in details in Section2 below.

[0110] As also illustrated in FIG. 8, the library of chimeric antibodyis screened in a yeast two hybrid system against the original antigenagainst which the non-human antibody is elicited. The selected chimericantibody is a chimeric Fab with non-human V_(H) and the rest of humanorigin.

[0111] Second, the chimeric heavy chain in the selected chimericantibody is humanized to form a fully human Fab with a human lightchain. As illustrated in FIG. 8, a library of human V_(H) (or CDRs) islinked to the human constant domain 1 of the heavy chain, CH1, in theyeast two-hybrid vector. The light chain of the selected chimericantibody from the first step is expressed from a separate expressioncassette and form a library of fully human Fabs by assembling with thelibrary of human heavy chains (V_(H)+C_(H)1). This library of fullyhuman Fabs is again subjected to a yeast two-hybrid screening againstthe The fully human Fab selected may be further linked to C_(H)2 andC_(H)3 at the C-terminus of the C_(H)1 domain, thereby resulting in afull length, fully human antibody.

[0112] 2. Screening the Library of Humanized Antibodies in Yeast

[0113] 1) Yeast Expression Vector

[0114] The library of humanized antibody sequences produced above can becloned into a yeast expression vector for expression and screening inyeast.

[0115] The yeast expression vector is based on a yeast plasmid,especially one from Saccharomyces cerevisiae. After transformation ofyeast cells, the exogenous DNA encoding humanized sequences are uptakenby the cells and subsequently expressed by the transformed cells.

[0116] More preferably, the expression vector may be a yeast-bacteriashuttle vector which can be propagated in either Escherichia coli oryeast Struhl, et al. (1979) Proc. Natl. Acad. Sci. 76:1035-1039. Theinclusion of E. coli plasmid DNA sequences, such as pBR322, facilitatesthe quantitative preparation of vector DNA in E. coli, and thus theefficient transformation of yeast.

[0117] The types of yeast plasmid vector that may serve as the shuttlemay be a replicating vector or an integrating vector. A replicatingvector is yeast vector that is capable of mediating its own maintenance,independent of the chromosomal DNA of yeast, by virtue of the presenceof a functional origin of DNA replication. An integrating vector reliesupon recombination with the chromosomal DNA to facilitate replicationand thus the continued maintenance of the recombinant DNA in the hostcell. A replicating vector may be a 2μ-based plasmid vector in which theorigin of DNA replication is derived from the endogenous 2μ plasmid ofyeast. Alternatively, the replicating vector may be an autonomouslyreplicating (ARS) vector, in which the “apparent” origin of replicationis derived from the chromosomal DNA of yeast. Optionally, thereplicating vector may be a centromeric (CEN) plasmid which carries inaddition to one of the above origins of DNA replication a sequence ofyeast chromosomal DNA known to harbor a centromere.

[0118] The vectors may be transformed into yeast cells in a closedcircular form or in a linear form. Transformation of yeast byintegrating vectors, although with inheritable stability, may not beefficient when the vector is in in a close circular form (e.g. 1-10transformants per ug of DNA). Linearized vectors, with free ends locatedin DNA sequences homologous with yeast chromosomal DNA, transforms yeastwith higher efficiency (100-1000 fold) and the transforming DNA isgenerally found integrated in sequences homologous to the site ofcleavage. Thus, by cleaving the vector DNA with a suitable restrictionendonuclease, it is possible to increase the efficiency oftransformation and target the site of chromosomal integration.Integrative transformation may be applicable to the genetic modificationof brewing yeast, providing that the efficiency of transformation issufficiently high and the target DNA sequence for integration is withina region that does not disrupt genes essential to the metabolism of thehost cell.

[0119] ARS plasmids, which have a high copy number (approximately 20-50copies per cell) (Hyman et al., 1982), tend to be the most unstable, andare lost at a frequency greater than 10% per generation. However, thestability of ARS plasmids can be enhanced by the attachment of acentromere; centromeric plasmids are present at 1 or 2 copies per celland are lost at only approximately 1% per generation.

[0120] In a preferred embodiment, the expression vector for expressingthe library of humanized antibody is based on the 2μ plasmid. The 2μplasmid is known to be nuclear in cellular location, but is inherited ina non-Mendelian fashion. Cells that lost the 2μ plasmid have been shownto arise from haploid yeast populations having an average copy number of50 copies of the 2μ plasmid per cell at a rate of between 0.001% and0.01% of the cells per generation. Futcher & Cox (1983) J. Bacteriol.154:612. Analysis of different strains of S. cerevisiae has shown thatthe plasmid is present in most strains of yeast including brewing yeast.The 2μ plasmid is ubiquitous and possesses a high degree of inheritablestability in nature.

[0121] The 2μ plasmid harbors a unique bidirectional origin of DNAreplication which is an essential component of all 2μ-based vectors. Theplasmid contains four genes, REP1, REP2, REP3 and FLP which are requiredfor the stable maintenance of high plasmid copy number per cell. Jaysramet al. (1983) Cell 34:95. The REP1 and REP2 genes encode transactingproteins which are believed to function in concert by interacting withthe REP3 locus to ensure the stable partitioning of the plasmid at celldivision. In this respect, the REP3 gene behaves as a cis acting locuswhich effects the stable segregation of the plasmid, and isphenotypically analogous to a chromosomal centromere. An importantfeature of the 2μ plasmid is the presence of two inverted DNA sequencerepeats (each 559 base-pairs in length) which separate the circularmolecule into two unique regions. Intramolecular recombination betweenthe inverted repeat sequences results in the inversion of one uniqueregion relative to the other and the production in vivo of a mixedpopulation of two structural isomers of the plasmid, designated A and B.Recombination between the two inverted repeats is mediated by theprotein product of a gene called the FLP gene, and the FLP protein iscapable of mediating high frequency recombination within the invertedrepeat region. This site specific recombination event is believed toprovide a mechanism which ensures the amplification of plasmid copynumber. Murray et al. (1987) EMBO J. 6:4205.

[0122] The expression vector may also contain an Escherichia coli originof replication and E. coli antibiotic resistance genes for propagationand antibiotic selection in bacteria. Many E. coli origins are known,including ColE1, pMB1 and pBR322, The ColE origin of replication ispreferably used in this invention. Many E. coli drug resistance genesare known, including the ampicillin resistance gene, thechloramphenoicol resistance gene and the tetracycline resistance gene.In one particular embodiment, the ampicillin resistance gene is used inthe vector.

[0123] The transformants that carry the humanized antibody sequences maybe selected by using various selection schemes. The selection istypically achieved by incorporating within the vector DNA a gene with adiscernible phenotype. In the case of vectors used to transformlaboratory yeast, prototrophic genes, such as LEU2, URA3 or TRP1, areusually used to complement auxotrophic lesions in the host. However, inorder to transform brewing yeast and other industrial yeasts, which arefrequently polyploid and do not display auxotrophic requirements, it isnecessary to utilize a selection system based upon a dominant selectablegene. In this respect replicating transformants carrying 2μ-basedplasmid vectors may be selected based on expression of marker geneswhich mediate resistance to: antibiotics such as G418, hygromycin B andchloramphenicol, or otherwise toxic materials such as the herbicidesulfometuron methyl, compactin and copper.

[0124] 2) Homologous Recombination in Yeast

[0125] The library of yeast expression vectors described above can beconstructed using a variety of recombinant DNA techniques. In apreferred embodiment, the library of yeast expression vectors containinga library of humanized antibody sequences are constructed by exploitingthe inherent ability of yeast cells to facilitate homologousrecombination at an extremely high efficiency. The mechanism ofhomologous recombination in yeast and its applications is brieflydescribed below.

[0126] Yeast Saccharomyces cerevisiae has an inherited genetic machineryto carry out efficient homologous recombination in the cell. Thismechanism is believed to benefit the yeast cells for chromosome repairpurpose and traditionally also called gap repair or gap filling. By thismechanism of efficient gap filling, mutations can be introduced intospecific loci of the yeast genome. For example, a vector carrying themutant gene contains two sequence segments that are homologous to the 5′and 3′ open reading frame (ORF) sequences of the gene that is intendedto be interrupted or mutated. The plasmid also contains a positiveselection marker such as a nutritional enzyme allele, such as ura3, oran antibiotic resistant marker such as Geneticine (g418) that areflanked by the two homologous segments. This plasmid is linearized andtransformed into the yeast cells. Through homologous recombinationbetween the plasmid and the yeast genome at the two homologousrecombination sites, a reciprocal exchange of the DNA content occursbetween the wild type gene in the yeast genome and the mutant gene(including the selection marker gene) that are flanked by the twohomologous sequence segments. By selecting for the positive nutritionalmarker, surviving yeast cells will loose the original wild type gene andwill adopt the mutant gene. Pearson B M, Hernando Y, and Schweizer M,(1998) Yeast 14: 391-399. This mechanism has also been used to makesystematic mutations in all 6,000 yeast genes or ORFs for functionalgenomics studies. Because the exchange is reciprocal, similar approachhas been used successfully for cloning yeast genomic fragments intoplasmid vector. Iwasaki T, Shirahige K, Yoshikawa H, and Ogasawara N,Gene 1991, 109 (1): 81-87.

[0127] By using homologous recombination in yeast, gene fragments orsynthetic oligonucleotides can also be cloned into a plasmid vectorwithout a ligation step. In this application, a targeted gene fragmentis usually obtained by PCR amplification (or by using the conventionalrestriction digestion out of an original cloning vector). Two shortfragment sequences that are homologous to the plasmid vector are addedto the 5′ and 3′ of the target gene fragment in the PCR amplification.This can be achieved by using a pair of PCR primers that incorporate theadded sequences. The plasmid vector typically includes a positiveselection marker such as nutritional enzyme allele such as ura3, or anantibiotic resistant marker such as geneticin (g418). The plasmid vectoris linearized by a unique restriction cut in between the sequencehomologies that are shared with the PCR-amplified target, therebycreating an artificial gap at the cleavage site. The linearized plasmidvector and the target gene fragment flanked by sequences homologous tothe plasmid vector are co-transformed into a yeast host strain. Theyeast recognizes the two stretches of sequence homologies between thevector and target fragment, and facilitates a reciprocal exchange of DNAcontents through homologous recombination at the gap. As theconsequence, the target fragment is automatically inserted into thevector without ligation in vitro.

[0128] There are a few factors that may influence the efficiency ofhomologous recombination in yeast. The efficiency of the gap repair iscorrelated with the length of the homologous sequences flanking both thelinearized vector and the targeted gene. Preferably, a minimum of 30base pairs may be required for the length of the homologous sequence,and 80 base pairs may give a near-optimized result. Hua, S. B. et al.(1997) “Minimum length of sequence homology required for in vitrocloning by homologous recombination in yeast” Plasmid 38:91-96. Inaddition, the reciprocal exchange between the vector and gene fragmentis strictly sequence-dependent, i.e. not causing frame shift in thistype of cloning. Therefore, such a unique characteristic of thegap-repair cloning assures insertion of gene fragments with both highefficiency and precision. The high efficiency makes it possible to clonetwo or three targeted gene fragments simultaneously into the same vectorin one transformation attempt. Raymond K., Pownder T. A., and Sexson S.L., (1999) Biotechniques 26: 134-141. The nature of precision sequenceconservation through homologous recombination makes it possible to clonetargeted genes in question into expression or fusion vectors for directfunction examinations. So far many functional or diagnostic applicationshave been reported using homologous recombination. El-Deiry W. W., etal., Nature Genetics1: 45-49, 1992 (for p53), and Ishioka C., et al.,PNAS, 94: 2449-2453, 1997 (for BRCA1 and APC).

[0129] A library of gene fragments may also be constructed in yeast byusing homologous recombination. For example, a human brain cDNA librarycan be constructed as a two-hybrid fusion library in vector pJG4-5.Guidotti E., and Zervos A. S. (1999) “In vivo construction of cDNAlibrary for use in the yeast two-hybrid systems” Yeast 15:715-720. Ithas been reported that a total of 6,000 pairs of PCR primers were usedfor amplification of 6,000 known yeast ORFs for a study of total yeastgenomic protein interaction. Hudson, J. Jr, et al. (1997) Genome Res.7:1169-1173. Uetz et al. conducted a comprehensive analysis ofprotein-protein interactions in Saccharomyces cerevisiae. Uetz et al.(2000) Nature 403:623-627. The protein-protein interaction map of thebudding yeast was studied by using a comprehensive system to examinetwo-hybrid interactions in all possible combinations between the yeastproteins. Ito et al. (2000) Proc. Natl. Acad. Sci. USA. 97:1143-1147.The genomic protein linkage map of Vaccinia virus was studied byMcCraith S., Holtzman T., Moss B., and Fields, S. (2000) Proc. Natl.Acad. Sci. USA 97: 4879-4884.

[0130] In a preferred embodiment, the library of humanized antibodysequences constructed in Section 1 is introduced into a yeast expressionvector by homologous recombination performed directly in yeast cells.The expression vector containing an AD domain may be any vectorengineered to carry the coding sequence of the AD domain.

[0131] According to this embodiment, the expression vector is preferablya yeast vector such as pGAD10 (Feiloter et al. (1994) “Construction ofan improved host strain for two hybrid screening” Nucleic Acids Res. 22:1502-1503), pACT2 (Harper et al (1993) “The p21 Cdk-interacting proteinCip1 is a protein inhibitor of G1 cyclin-dependent kinase” Cell75:805-816), and pGADT7 (“Matchmaker Gal4 two hybrid system 3 andlibraries user manual” (1999), Clontech PT3247-1, supplied by Clontech,Palo Alto, Calif.).

[0132] Also according to this embodiment, the flanking sequences thatare added to the 5′ and 3′-terminus of scFv sequences (or each of theheavy chain and light chain for the double chain approach) in thelibrary. The flanking sequence is preferably between about 30-120 bp inlength, more preferably between about 40-90 bp in length, and mostpreferably between about 45-55 bp in length.

[0133] When the library of humanized antibody sequences is inserted intoan expression vector containing an AD domain, it is preferred that thereading frame of the humanized antibody sequence is conserved withupstream AD reading frame.

[0134] Depending on the cloning expression vector used, additionalfeatures such as affinity tags and unique restriction enzyme recognitionsites may be added to the expression for the convenience of detectionand purification of the inserted humanized antibody sequences. Examplesof affinity tags include, but are not limited to, a polyhistidine tract,polyarginine, glutathione-S-transferase (GST), maltose binding protein(MBP), a portion of staphylococcal protein A (SPA), and variousimmunoaffinity tags (e.g. protein A) and epitope tags such as thoserecognized by the EE (Glu-Glu) antipeptide antibodies.

[0135] Optionally, expression of the library of humanized antibodysequences may be under the transcriptional control of an induciblepromoter. One example of such an expression vector is available fromClontech, pBridge® (catalog No. 6184-1). The expression vector,pBridge®, contains one expression unit that controls expression of a Gal4 BD domain and another expression unit that includes an induciblepromoter Pmat25. Tirode, E. et al. (1997) J. Biol. Chem.272:22995-22999.

[0136] The linearized vector DNA may be mixed with equal or excessamount of the inserts of humanized antibody sequences generated inSection 1. The linearized vector DNA and the inserts are co-transformedinto host cells, such as competent yeast cells. Recombinant clones maybe selected based on survival of cells in a nutritional selection mediumor based on other phenotypic markers. Either the linearized vector orthe insert alone may be used as a control for determining the efficiencyof recombination and transformation.

[0137] Other homologous recombination systems may be used to generatethe library of expression vectors of the present invention. For example,the recombination between the library of humanized antibody sequencesand the recipient expression vector may be facilitated by site-specificrecombination.

[0138] The site-specific recombination employs a site-specificrecombinase, an enzyme which catalyzes the exchange of DNA segments atspecific recombination sites. Site-specific recombinases present in someviruses and bacteria, and have been characterized to have bothendonuclease and ligase properties. These recombinases, along withassociated proteins in some cases, recognize specific sequences of basesin DNA and exchange the DNA segments flanking those segments. Landy, A.(1993) Current Opinion in Biotechnology 3:699-707.

[0139] A typical site-specific recombinase is CRE recombinase. CRE is a38-kDa product of the cre (cyclization recombination) gene ofbacteriophage P1 and is a site-specific DNA recombinase of the Intfamily. Sternberg, N. et al. (1986) J. Mol. Biol. 187: 197-212. CRErecognizes a 34-bp site on the P1 genome called loxP (locus of X-over ofP1) and efficiently catalyzes reciprocal conservative DNA recombinationbetween pairs of loxP sites. The loxP site [SEQ ID NO: 1] consists oftwo 13-bp inverted repeats flanking an 8-bp nonpalindromic core region.CRE-mediated recombination between two directly repeated loxP sitesresults in excision of DNA between them as a covalently closed circle.Cre-mediated recombination between pairs of loxP sites in invertedorientation will result in inversion of the intervening DNA rather thanexcision. Breaking and joining of DNA is confined to discrete positionswithin the core region and proceeds on strand at a time by way oftransient phophotyrosine DNA-protein linkage with the enzyme.

[0140] The CRE recombinase also recognizes a number of variant or mutantlox sites relative to the loxP sequence. Examples of these Crerecombination sites include, but are not limited to, the loxB, loxL andloxR sites which are found in the E. coli chromosome. Hoess et al.(1986) Nucleic Acid Res. 14:2287-2300. Other variant lox sites include,but are not limited to, loxB, loxL, loxR, loxP3, loxP23, loxΔ86,loxΔ117, loxP511, and loxC2.

[0141] Examples of the non-CRE recombinases include, but are not limitedto, site-specific recombinases include: att sites recognized by the Intrecombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL,and attR), the FRT sites recognized by FLP recombinase of the 2 piplasmid of Saccharomyces cerevisiae, the recombination sites recognizedby the resolvase family, and the recombination site recognized bytransposase of Bacillus thruingiensis.

[0142] Subsequent analysis may also be carried out to determine theefficiency of homologous recombination that results in correct insertionof the humanized antibody sequences into the expression vector. Forexample, PCR amplification of the inserts of the humanized antibodysequences directly from the selected yeast clone may reveal how manyclones are recombinant. Libraries with minimum of 90% recombinant clonesare preferred. The same PCR amplification of selected clones may alsoreveal the insert size. Although a small fraction of the library maycontain double or triple inserts, the majority (>90%) is preferably tohave a single insert with the expected size.

[0143] To verify sequence diversity of the inserts in the selectedclones, PCR amplification product with the correct size of insert may befingerprinted with frequent digesting restriction enzymes. From a gelelectrophoresis pattern, it may be determined whether the clonesanalyzed are of the same identity or of the distinct or diversifiedidentity. The PCR products may also be sequenced directly to reveal theidentity of inserts and the fidelity of the cloning procedure and toprove the independence and diversity of the clones.

[0144] 3) Yeast Two-hybrid Screening

[0145] The present invention also provides methods for screening alibrary of humanized antibody against a target antigen. The targetantigen may be the original antigen against which the non-human antibodyis elicited. In this case, the humanized antibody selected is truly“humanized” from the original non-human antibody. Alternatively, thetarget antigen against which the library of humanized antibody isscreened may be an antigen different from the original antigen. Forexample, the antigen may be an isoform in the same family of proteins asthe original antigen. Through this process, a humanized antibody withhigh binding affinity to a new target antigen can be selected withoutfirst obtaining a non-human antibody against this new target antigen.

[0146] The library of humanized antibody is screened against the targetantigen in a yeast two-hybrid system. The two-hybrid system is aselection scheme designed to screen for polypeptide sequences which bindto a predetermined polypeptide sequence present in a fusion protein.Chien et al. (1991) Proc. Natl. Acad. Sci. (USA) 88: 9578). Thisapproach identifies protein-protein interactions in vivo throughreconstitution of a transcriptional activator. Fields and Song (1989)Nature 340: 245), the yeast Gal 4 transcription protein. The method isbased on the properties of the yeast Gal 4 protein, which consists ofseparable domains responsible for DNA-binding and transcriptionalactivation. Polynucleotides encoding two hybrid proteins, one consistingof the yeast Gal 4 DNA-binding domain (BD) fused to a polypeptidesequence of a known protein and the other consisting of the Gal4activation domain (AD) fused to a polypeptide sequence of a secondprotein, are constructed and introduced into a yeast host cell.Intermolecular binding between the two fusion proteins reconstitutes theGal4 DNA-binding domain with the Gal4 activation domain, which leads tothe transcriptional activation of a reporter gene (e.g., lacZ, HIS3)which is operably linked to a Gal4 binding site.

[0147] Typically, the two-hybrid method is used to identify novelpolypeptide sequences which interact with a known protein. Silver andHunt (1993) Mol. Biol. Rep. 17: 155; Durfee et al. (1993) Genes Devel.7; 555; Yang et al. (1992) Science 257: 680; Luban et al. (1993) Cell73: 1067; Hardy et al. (1992) Genes Devel. 6; 801; Bartel et al. (1993)Biotechniques 14: 920; and Vojtek et al. (1993) Cell 74: 205. Thetwo-hybrid system was used to detect interactions between three specificsingle-chain variable fragments (scFv) and a specific antigen. De Jaegeret al. (2000) FEBS Lett. 467:316-320. The two-hybrid system was alsoused to screen against cell surface proteins or receptors such asreceptors of hematopoietic super family in yeast. Ozenberger, B. A., andYoung, K. H. (1995) “Functional interaction of ligands and receptors ofhematopoietic superfamily in yeast” Mol Endocrinol. 9:1321-1329.

[0148] Variations of the two-hybrid method have been used to identifymutations of a known protein that affect its binding to a second knownprotein Li and Fields (1993) FASEB J. 7: 957; Lalo et al. (1993) Proc.Natl. Acad. Sci. (USA) 90: 5524; Jackson et al. (1993) Mol. Cell. Biol.13; 2899; and Madura et al. (1993) J. Biol. Chem. 268: 12046.

[0149] Two-hybrid systems have also been used to identify interactingstructural domains of two known proteins or domains responsible foroligomerization of a single protein. Bardwell et al. (1993) Med.Microbiol. 8: 1177; Chakraborty et al. (1992) J. Biol. Chem. 267: 17498;Staudinger et al. (1993) J. Biol. Chem. 268: 4608; and Milne G T; WeaverD T (1993) Genes Devel. 7; 1755; Iwabuchi et al. (1993) Oncogene 8;1693; Bogerd et al. (1993) J. Virol. 67: 5030).

[0150] Variations of two-hybrid systems have been used to study the invivo activity of a proteolytic enzyme. Dasmahapatra et al. (1992) Proc.Natl. Acad. Sci. (USA) 89: 4159. Alternatively, an E. coli/BCCPinteractive screening system was used to identify interacting proteinsequences (i.e., protein sequences which heterodimerize or form higherorder heteromultimers). Germino et al. (1993) Proc. Natl. Acad. Sci.(U.S.A.) 90: 933; and Guarente L (1993) Proc. Natl. Acad. Sci. (U.S.A.)90: 1639.

[0151] Typically, selection of binding protein using a two-hybrid methodrelies upon a positive association between two Gal4 fusion proteins,thereby reconstituting a functional Gal4 transcriptional activator whichthen induces transcription of a reporter gene operably linked to a Gal4binding site. Transcription of the reporter gene produces a positivereadout, typically manifested either (1) as an enzyme activity (e.g.,β-galactosidase) that can be identified by a colorimetric enzyme assayor (2) as enhanced cell growth on a defined medium (e.g., HIS3 and Ade2). Thus, the method is suited for identifying a positive interaction ofpolypeptide sequences, such as antibody-antigen interactions.

[0152] False positives clones that indicate activation of the reportergene irrespective of the specific interaction between the two hybridproteins, may arise in the two-hybrid screening. Various procedures havedeveloped to reduce and eliminate the false positive clones from thefinal positives. For example, 1) prescreening the clones that containsthe target vector and shows positive in the absence of the two-hybridpartner (Bartel, P. L., et al. (1993) “Elimination of false positivesthat arise in using the two-hybrid system” BioTechniques 14:920-924); 2)by using multiple reporters such as His3, β-galactosidase, and Ade2(James, P. et al. (1996) “Genomic libraries and a host strain designedfor highly efficient two-hybrid selection in yeast” Genetics144:1425-1436); 3) by using multiple reporters each of which is underdifferent GAL 4-responsive promoters such as those in yeast strain Y190where each of the His 3 and β-Gal reporters is under the control of adifferent promoter Gal 1 or Gal 10, but both response to Gal 4 signaling(Durfee, T., et al (1993) “The retinoblastoma protein associates withthe protein phosphatase type 1 catalytic subunit” Genes Devel.7:555-569); and 4) by post-screening assays such as testing isolateswith target consisting of GAL 4-BD alone.

[0153] In addition, the false positive clones may also be eliminated byusing unrelated targets to confirm specificity. This is a standardcontrol procedure in the two-hybrid system which can be performed afterthe library isolate is confirmed by the above-described 1)-4)procedures. Typically, the library clones are confirmed byco-transforming the initially isolated library clones back into theyeast reporter strain with one or more control targets unrelated to thetarget used in the original screening. Selection is conducted toeliminate those library clones that show positive activation of thereporter gene and thus indicate non-specfic interactions with multiple,related proteins.

[0154] The present invention provides efficient methods for screening alibrary of humanized antibody contained in a library of expressionvectors for their affinity binding to a specific antigen.

[0155] According to the present invention, the method comprises:

[0156] expressing a library of humanized antibodies in yeast cells;

[0157] expressing a specific target protein in the yeast cellsexpressing the humanized antibodies; and

[0158] selecting those yeast cells in which a reporter gene isexpressed, the expression of the reporter gene being activated bybinding of the humanized antibody to the target protein.

[0159] According to the method, the diversity of the library ofhumanized antibody is preferably between 10²-10⁸, more preferablybetween 10⁴-10⁸, and most preferably between 10⁵-10⁸.

[0160] According to the embodiment, the target protein is expressed as afusion and screened against the library of humanized antibody. Thus, thestep of expressing the library of humanized antibody may includetransforming a library of expression vectors encoding the library ofhumanized antibody into the yeast cells which contain a reporterconstruct comprising the reporter gene. The report gene expression isunder transcriptional control of a transcription activator comprising anactivation domain and a DNA binding domain.

[0161] Each of the expression vectors comprises a humanized antibodysequence (e.g., scFv, heavy chain or light chain) fused with either theactivation domain or the DNA binding domain of the transcriptionactivator.

[0162] Optionally, the step of expressing the target protein includestransforming a target expression vector into the yeast cellssimultaneously or sequentially with the library of humanized expressionvectors encoding humanized antibody. The target expression vectorcomprises a second transcription sequence encoding either the activationdomain AD or the DNA binding domain BD of the transcription activatorwhich is not expressed by the library of humanized antibody expressionvectors; and a target sequence encoding the target protein or peptide.

[0163]FIG. 9 illustrates a flow diagram of a preferred embodiment of theabove described method. As illustrated in FIG. 9, the sequence librarycontaining scFv is fused with an AD domain upstream, the AD-scFvvectors. The coding sequence of the target protein (labeled as “Target”)is contained in another expression vector and fused with a BD domain,forming the BD-Target vector.

[0164] The AD-scFv vector and the BD-Target vector may be co-transformedinto a yeast cell by using method known in the art. Gietz, D. et al.(1992) “Improved method for high efficiency transformation of intactyeast cells” Nucleic Acids Res. 20:1425. The construct carrying thespecific DNA binding site and the reporter gene (labeled as “Reporter”)may be stably integrated into the genome of the host cell or transientlytransformed into the host cell. Upon expression of the sequences in theexpression vectors, the library of protein complexes comprising AD-scFv,undergo protein folding in the host cell and adopt variousconformations. Some of the AD-scFv fusion protein complexes may bind tothe Target protein expressed by the BD-Target vector in the host cell,thereby bringing the AD and BD domains to a close proximity in thepromoter region (i.e., the specific DNA binding site) of the reporterconstruct and thus reconstituting a functional transcription activatorcomposed of the AD and BD domains. As a result, the AD activates thetranscription of the reporter gene downstream from the specific DNAbinding site, resulting in expression of the reporter gene, such as thelacZ reporter gene. Clones showing the phenotype of the reporter geneexpression are selected, and the AD-scFv vectors are isolated. Thecoding sequences for scFv are identified and characterized.

[0165] Alternatively, the steps of expressing the library of humanizedantibody and expressing the target fusion protein includes causingmating between first and second populations of haploid yeast cells ofopposite mating types.

[0166] The first population of haploid yeast cells comprises a libraryof expression vectors encoding the library of humanized antibody. Eachof the expression vector comprises a first transcription sequenceencoding either the activation domain AD or the DNA binding domain BD ofthe transcription activator and a scFv encoding an humanized antibody.

[0167] The second population of haploid yeast cells comprises a targetexpression vector. The target expression vector comprises a secondtranscription sequence encoding either the activation domain AD or theDNA binding domain BD of the transcription activator which is notexpressed by the library of tester expression vectors; and a targetsequence encoding the target protein or peptide. Either the first orsecond population of haploid yeast cells comprises a reporter constructcomprising the reporter gene whose expression is under transcriptionalcontrol of the transcription activator.

[0168] In this method, the haploid yeast cells of opposite mating typesmay preferably be α and a type strains of yeast. The mating between thefirst and second populations of haploid yeast cells of α and a-typestrains may be conducted in a rich nutritional culture medium.

[0169]FIG. 10 illustrates a flow diagram of a preferred embodiment ofthe above described method. As illustrated in FIG. 10, the sequencelibrary containing a scFv fused with an AD domain upstream, the AD-scFvvectors. The library of the AD-scFv vectors are transformed into haploidyeast cells such as the a type strain of yeast.

[0170] The coding sequence of the target protein (labeled as “Target”)is contained in another expression vector and fused with a BD domain,forming the BD-Target vector. The BD-Target vector is transformed intohaploid cells of opposite mating type of the haploid cells containingthe AD-scFv vectors, such as the α type strain of yeast. The constructcarrying the specific DNA binding site and the reporter gene (labeled as“Reporter”) may be transformed into the haploid cells of either the typea or type α strain of yeast.

[0171] The haploid cells of the type a and type α strains of yeast aremated under suitable conditions such as low speed of shaking in liquidculture, physical contact in solid medium culture, and rich medium suchas YPD. Bendixen, C. et al. (1994) “A yeast mating-selection scheme fordetection of protein-protein interactions”, Nucleic Acids Res. 22:1778-1779. Finley,Jr., R. L. & Brent, R. (1994) “Interaction matingreveals lineary and ternery connections between Drosophila cell cycleregulators”, Proc. Natl. Acad. Sci. USA, 91:12980-12984. As a result,the AD-scFv, the BD-Target expression vectors and the Reporter constructare taken into the parental diploid cells of the a and type α strain ofhaploid yeast cells.

[0172] Upon expression of the sequences in the expression vectors in theparental diploid cells, the library of protein complexs formed betweenAD-scFv, labeled as the AD-scFv fusion protein, undergo protein foldingin the host cell and adopt various conformations. Some of the AD-scFvprotein complexes may bind to the Target protein expressed by theBD-Target vector in the parental diploid cell, thereby bringing the ADand BD domains to a close proximity in the promoter region (i.e., thespecific DNA binding site) of the reporter construct and thusreconstituting a functional transcription activator composed of the ADand BD domains. As a result, the AD activates the transcription of thereporter gene downstream from the specific DNA binding site, resultingin expression of the reporter gene, such as the lacZ reporter gene.Clones showing the phenotype of the reporter gene expression areselected, and the AD-scFv vectors are isolated. The coding sequences forscFv are identified and characterized.

[0173] A wide variety of reporter genes may be used in the presentinvention. Examples of proteins encoded by reporter genes include, butare not limited to, easily assayed enzymes such as β-galactosidase,α-galactosidase, luciferase, β-glucuronidase, chloramphenicol acetyltransferase (CAT), secreted embryonic alkaline phosphatase (SEAP),fluorescent proteins such as green fluorescent protein (GFP), enhancedblue fluorescent protein (EBFP), enhanced yellow fluorescent protein(EYFP) and enhanced cyan fluorescent protein (ECFP); and proteins forwhich immunoassays are readily available such as hormones and cytokines.The expression of these reporter genes can also be monitored bymeasuring levels of mRNA transcribed from these genes.

[0174] When the screening of the humanized antibody library is conductedin yeast cells, certain reporter(s) are of nutritional reporter whichallows the yeast to grow on the specific selection medium plate. This isa very powerful screening process, as has been shown by many publishedpapers. Examples of the nutritional reporter include, but are notlimited to, His3, Ade2, Leu2, Ura3, Trp1 and Lys2. The His3 reporter isdescribed in Bartel, P. L. et al. (1993) “Using the two-hybrid system todetect protein-protein interactions”, in Cellular interactions inDevelopment: A practical approach, ed. Hastley, D. A., Oxford Press,pages 153-179. The Ade2 reporter is described in Jarves, P. et al.(1996) “Genomic libraries and a host strain designed for highlyefficient two-hybrid selection in yeast” Genetics 144:1425-1436.

[0175] For example, the library of humanized antibody expression vectorsmay be transformed into haploid cells of the α mating type of yeaststrain. The plasmid containing the sequence encoding the target proteinfused with a BD domain is transformed into haploid cells of the a matingtype of yeast strain.

[0176] Equal volume of AD-scFv library-containing yeast stain (α-type)and the BD-target-containing yeast strain (a-type) are inoculated intoselection liquid medium and incubated separately first. These twocultures are then mixed and allowed to grow in rich medium such as 1×YPDand 2×YPD. Under the rich nutritional culture condition, the two haploidyeast strains will mate and form diploid cells. At the end of thismating process, these yeast cells are plated into selection plates. Amultiple-marker selection scheme may be used to select yeast clones thatshow positive interaction between the antibodies in the library and thetarget. For example, a scheme of SD/-Leu-Trp-His-Ade may be used. Thefirst two selections (Leu-Trp) are for markers (Leu and Trp) expressedfrom the AD-Antibody library and the BD-Target vector, respectively.Through this dual-marker selection, diploid cells retaining both BD andAD vectors in the same yeast cells are selected. The latter two markers,His-Ade, are used to screen for those clones that express the reportergene from parental strain, presumably due to affinity binding betweenthe antibodies in the library and the target.

[0177] After the screening by co-transformation, or by mating screeningas described above, the putative interaction between the target antigenwith the humanized antibody encoded by the library clone isolates can befurther tested and confirmed in vitro or in vivo.

[0178] In vitro binding assays may be used to confirm the positiveinteraction between the humanized expressed by the clone isolate and thetarget protein or peptide (e.g. the target antigen). For example, the invitro binding assay may be a “pull-down” method, such as using GST(glutathione S-transferase)-fused target antigen as matrix-bindingprotein, and with in vitro expressed library clone isolate that arelabeled with a radioactive or non-radioactive group. While the targetantigen is bound to the matrix through GST affinity substrate(glutathione-agarose), the library clone isolate will also bind to thematrix through its affinity with the target antigen. The in vitrobinding assay may also be a co-immuno-precipitation (Co-IP) method usingtwo affinity tag antibodies. In this assay, both the target antigen andthe library clone isolate are in vitro expressed fused with peptidetags, such as HA (haemaglutinin A) or Myc tags. The gene probe is firstimmuno-precipitated with an antibody against the affinity peptide tag(such as HA) that the target gene probe is fused with. Then the secondantibody against a different affinity tag (such as Myc) that is fusedwith the library clone isolate is used for reprobing the precipitate.

[0179] In vivo assays may also be used to confirm the positiveinteraction between the humanized antibody expressed by the cloneisolate and the target antigen. For example, a mammalian two-hybridsystem may serve as a reliable verification system for the yeasttwo-hybrid library screening. In this system, the target antigen and thelibrary clone are fused with Gal 4 DNA-binding domain or a mammalianactivation domain (such as VP-16) respectively. These two fusionproteins under control of a strong and constitutive mammalian promoter(such as CMV promoter) are introduced into mammalian cells bytransfection along with a reporter responsive to Gal 4. The reporter canbe CAT gene (chloramphenical acetate transferase) or other commonly usedreporters. After 2-3 days of transfection, CAT assay or other standardassays will be performed to measure the strength of the reporter whichis correlated with the strength of interaction between the targetantigen and the library clone isolate.

[0180] According to the present invention, other yeast two-hybridsystems may be employed, including but not limited to SOS-RAS system(SRS), Ras recruitment system (RRS), and ubiquitin split system.Brachmann and Boeke (1997) “Tag games in yeast: the two-hybrid systemand beyond” Current Opinion Biotech. 8:561-568. In thesenon-conventional yeast two-hybrid systems, the first or secondpolypeptide subunit may further comprise a signaling domain forscreening the library of the protein complexes based thesenon-conventional two-hybrid methods. Examples of such signaling domainincludes but are not limited to a Ras guanyl nucleotide exchange factor(e.g. human SOS factor), a membrane targeting signal such as amyristoylation sequence and farnesylation sequence, mammalian Raslacking the carboxy-terminal domain (the CAAX box), and a ubiquitinsequence.

[0181] SRS and RRS systems are alternative two-hybrid systems forstudying protein-protein interaction in cytoplasm. Both systems use ayeast strain with temperature-sensitive mutation in the cdc25 gene, theyeast homologue of human Sos (hSos). This protein, a guanyl nucleotideexchange factor, binds and activates Ras, that triggers the Rassignaling pathway. The mutation in the cdc25 protein is temperaturesensitive; the cells can grow at 25° C. but not at 37° C. In the SRSsystem, this cdc25 mutation is complemented by the hSos gene product toallow growth at 37° C., providing that the hSos protein is localized tothe membrane via a protein-protein interaction (Aronheim et al. 1997,Mol. Cel. Biol. 17:3094-3102). In the RRS system, the mutation iscomplemented by a mammalian activated Ras with its CAAX box at itscarboxy terminus upon recruitment to the plasma membrane viaprotein-protein interaction (Broder et al, 1998, Current Biol.8:1121-1124).

[0182] 3. Screening of a Library of Humanized Antibody by RibosomeDisplay

[0183] The present invention also provides methods for screening alibrary of humanized antibody against a specific target antigen viaribosome display in vitro.

[0184] Ribosome display is a form of protein display for in vitroselection against a target ligand. In this system, mRNA encoding thetester protein (e.g. an antibody) and the translated tester protein areassociated through the ribosome complex, also called anantibody-ribosome-mRNA (ARM) complex. He and Taussig (1997) Nucleic AcidResearch 25:5132-5134. The principle behind this approach is that singlechain antibody can be functionally produced in an in vitro translationsystem (e.g. rabbit reticulocyte lysate), and in the absence of a stopcodon, individual nascent proteins remain associated with theircorresponding mRNa as stable ternary polypeptide-ribosome-mRNA complexesin such a cell-free system.

[0185]FIG. 11 illustrates a method of the present invention used forscreening the library of humanized antibody sequences constructed inSection 1 in the ARM system. As illustrated in FIG. 11, each member ofthe library of humanized antibody sequences for ribosome displayincludes a bacterial phage T7 promoter and protein synthesis initiationsequence attached to the 5′ end of the cDNA encoding the antibody (e.g.,scFv, V_(H) or V_(L)) and no stop codon in the 3′ end. Because the cDNApool is depleted of the stop codon, when the mRNA is transcribed fromthe cDNA and is subject to in vitro translation, the mRNA will still beattached to the ribosome and mRNA, forming the ARM complex. The libraryof humanized antibody that is translated from the cDNA gene pool anddisplayed on the surface of the ribosome can be screened against aspecific target antigen. The in vitro transcription and translation ofthis library may be carried out in rabbit reticulocyte lysate in thepresence of methionine at 30° C. by using the commercially availablesystems, such as TNT T7 Quick Coupled Transcription/Translation System(Promega, Madison, Wis.).

[0186] The specific target antigen may be any molecule, including, butnot limited to, biomacromolecules such as protein, DNA, RNA,polycarbohydrate or small molecules such as peptide, organic compoundand organometallic complexes. Preferably, the target antigen isimmobilized to a solid substrate, such as a chromatography resin bycovalent linkage to enrich for those ribosomes with high affinityhumanized antibody attached. By affinity chromatography, the ribosomeswith high affinity humanized antibody attached are isolated. The mRNAencoding the high affinity humanized antibody is recovered from theisolated ARM complexes and subject to reverse transcriptase (RT)/PCR tosynthesize and amplify the cDNA of the selected antibody. This completesthe first cycle of the panning process for antibody isolation and itscoding sequence characterization.

[0187] Such a panning process may be repeated until humanized antibodywith desirably affinity is isolated. Specifically, the sequence encodingthe selected humanized antibody in the first cycle may be mutagenized togenerate a secondary library of humanized antibody sequences which aresubject to another cycle of ribosome display panning. The mutagenesismay be carried out simultaneously in the RT/PCR step, which not onlysynthesizes the cDNA but also mutagenizes the cDNA randomly, e.g., byerror-prone PCR. This secondary library of humanized antibody sequencesare then transcribed and translated in vitro following similar steps forthe first round of selection. The library of humanized antibodydisplayed on the ARM complexes are subject to the second round ofscreening against the same target antigen to select for humanizedantibody with higher affinity than the one(s) selected from the firstround of selection. The whole panning process can be reiterated toproduce humanized antibody with perhaps much higher affinity than theoriginal non-human antibody from which the first library of humanizedantibody is derived.

[0188] 4. Screening of a Library of Humanized Antibody by mRNA Display

[0189] The present invention also provides methods for screening alibrary of humanized antibody against a specific target antigen via mRNAdisplay in vitro.

[0190] Similar to ribosome display described above, mRNA display is aform of protein display for in vitro selection against a target ligand.In this system, mRNA encoding the tester protein (e.g. an antibody) andthe translated tester protein are associated through covalent linkage.Keefe and Szostak (2001) “Functional proteins from a random-sequencelibrary” Nature 410:715-718; Wilson et al. (2001) “The use of mRNAdisplay to select high-affinity protein-binding peptides” Proc Natl AcadSci U S A 98:3750-3755; Cho et al. (2000) “Constructing high complexitysynthetic libraries of long ORFs using in vitro selection” J Mol Biol.297:309-319; and Roberts and Szostak (1997) “RNA-peptide fusions for thein vitro selection of peptides and proteins” Proc Natl Acad Sci U S A.94:12297-12302. The in vitro translated protein is covalently linked atits C-terminus to the 3′ end of its encoding mRNA by a peptidyl acceptorlinker such as the antibiotic puromycin. Specifically, in thetranslation reaction, puromycin enters the “A” site of ribosomes andforms a covalent bond with the nascent peptide at the C-terminus. Such acovalently associated mRNA-protein complex can be selected for itsbinding affinity toward a target ligand in vitro. After RT-PCR cDNAencoding the binding protein can be amplified and identified.

[0191]FIG. 12 illustrates a method of the present invention used forscreening the library of humanized antibody sequences constructed inSection 1 via mRNA display. As illustrated in FIG. 12, each member ofthe library of humanized antibody sequences for mRNA display includes abacterial phage T7 promoter and protein synthesis initiation sequenceattached to the 5′ end of the cDNA encoding the antibody (e.g., scFv,V_(H) or V_(L)) and no stop codon in the 3′ end. A peptidyl acceptorlinker such as puromycin is added to the in vitro transcribed mRNAlibrary to react with the 3′-end of the mRNA. The in vitro transcriptionand translation of this library may be carried out in rabbitreticulocyte lysate in the presence of methionine at 30° C. by using thecommercially available systems, such as TNT T7 Quick CoupledTranscription/Translation System (Promega, Madison, Wis.). The nascentprotein translated from the 3′-end modified mRNA pool reacts withpuromycin at its C-terminus to form the covalently bound mRNA-antibodycomplex.

[0192] Still referring to FIG. 12, the library of antibody displayedlinked to its encoding mRNA can be screened against a specific targetantigen.

[0193] The specific target antigen may be any molecule, including, butnot limited to, biomacromolecules such as protein, DNA, RNA,polycarbohydrate or small molecules such as peptide, organic compoundand organometallic complexes. Preferably, the target antigen isimmobilized to a solid substrate, such as a chromatography resin bycovalent linkage to enrich for those ribosomes with high affinityhumanized antibody attached. By affinity chromatography, themRNA-antibody complexes with high affinity toward the target antigen areisolated. The mRNA encoding the high affinity humanized antibody isrecovered from the isolated mRNA-Antibody complexes and subject toreverse transcriptase (RT)/PCR to synthesize and amplify the cDNA of theselected antibody. This completes the first cycle of the panning processfor antibody isolation and its coding sequence characterization.

[0194] Such a panning process may be repeated until humanized antibodywith desirably affinity is isolated. Specifically, the sequence encodingthe selected humanized antibody in the first cycle may be mutagenized togenerate a secondary library of humanized antibody sequences which aresubject to another cycle of ribosome display panning. The mutagenesismay be carried out simultaneously in the RT/PCR step, which not onlysynthesizes the cDNA but also mutagenizes the cDNA randomly, e.g., byerror-prone PCR. This secondary library of humanized antibody sequencesare then transcribed and translated in vitro following similar steps forthe first round of selection. The library of humanized antibodydisplayed on the mRNA-Antibody complexes are subject to the second roundof screening against the same target antigen to select for humanizedantibody with higher affinity than the one(s) selected from the firstround of selection. The whole panning process can be reiterated toproduce humanized antibody with perhaps much higher affinity than theoriginal non-human antibody from which the first library of humanizedantibody is derived.

[0195] 5. Mutagenesis of the Humanized Antibody Leads PositivelySelected Against a Target Antigen—Affinity Maturation

[0196] As described above, humanized antibody leads, such as scFv ordsFv, can be identified through selection of the primary librarycarrying humanized antibody against a specific target antigen. Thecoding sequences of these humanized antibody leads may be mutagenized invitro or in vivo to generated a secondary library more diverse thanthese leads. The mutagenized leads can be selected against the targetantigen again in vivo following similar procedures described for theselection of the primary library carrying humanized antibody. Suchmutagenesis and selection of primary humanized antibody leadseffectively mimics the affinity maturation process naturally occurringin a mammal that produces antibody with progressive increase in theaffinity to the immunizing antigen.

[0197] The coding sequences of the humanized antibody leads may bemutagenized by using a wide variety of methods. Examples of methods ofmutagenesis include, but are not limited to site-directed mutagenesis,error-prone PCR mutagenesis, cassette mutagenesis, random PCRmutagenesis, DNA shuffling, and chain shuffling.

[0198] Site-directed mutagenesis or point mutagenesis may be used togradually the humanized antibody sequences in specific regions. This isgenerally accomplished by using oligonucleotide-directed mutagenesis.For example, a short sequence of an antibody lead may be replaced with asynthetically mutagenized oligonucleotide in either the heavy chain orlight chain region or both. The method may not be efficient formutagenizing large numbers of humanized antibody sequences, but may beused for fine toning of a particular lead to achieve higher affinitytoward a specific target protein.

[0199] Cassette mutagenesis may also be used to mutagenize the humanizedantibody sequences in specific regions. In a typical cassettemutagenesis, a sequence block, or a region, of a single template isreplaced by a completely or partially randomized sequence. However, themaximum information content that can be obtained may be statisticallylimited by the number of random sequences of the oligonucleotides.Similar to point mutagenesis, this method may also be used for finetoning of a particular lead to achieve higher affinity toward a specifictarget protein.

[0200] Error-prone PCR, or “poison” PCR, may be used to the humanizedantibody sequences by following protocols described in Caldwell andJoyce (1992) PCR Methods and Applications 2:28-33. Leung, D. W. et al.(1989) Technique 1:11-15. Shafikhani, S. et al. (1997) Biotechniques23:304-306. Stemmer, W. P. et al. (1994) Proc. Natl. Acad. Sci. USA91:10747-10751.

[0201]FIG. 13 illustrates an example of the method of the presentinvention for affinity maturation of humanized antibody leads selectedfrom the primary antibody library. As illustrated in FIG. 13, the codingsequences of the humanized antibody leads selected from clonescontaining the primary library are mutagenized by using a poison PCRmethod. Since the coding sequences of the humanized antibody library arecontained in the expression vectors isolated from the selected clones,one or more pairs of PCR primers may be used to specifically amplify theV_(H) and V_(L) region out of the vector. The PCR fragments containingthe V_(H) and V_(L) sequences are mutagenized by the poison PCR underconditions that favors incorporation of mutations into the product.

[0202] Such conditions for poison PCR may include a) high concentrationsof Mn²⁺ (e.g. 0.4-0.6 mM) that efficiently induces malfunction of TaqDNA polymerase; and b) disproportionally high concentration of onenucleotide substrate (e.g., dGTP) in the PCR reaction that causesincorrect incorporation of this high concentration substrate into thetemplate and produce mutations. Additionally, other factors such as, thenumber of PCR cycles, the species of DNA polymerase used, and the lengthof the template, may affect the rate of mis-incorporation of “wrong”nucleotides into the PCR product. Commercially available kits may beutilized for the mutagenesis of the selected antibody library, such asthe “Diversity PCR random mutagenesis kit” (catalog No. K1830-1,Clontech, Palo Alto, Calif.).

[0203] The PCR primer pairs used in mutagenesis PCR may preferablyinclude regions matched with the homologous recombination sites in theexpression vectors. This design allows re-introduction of the PCRproducts after mutagenesis back into the yeast host strain again viahomologous recombination. This also allows the modified V_(H) or V_(L)region to be fused with the AD domain directly in the expression vectorin the yeast.

[0204] Still referring to FIG. 13, the mutagenized scFv fragments areinserted into the expression vector containing an AD domain viahomologous recombination in haploid cells of α type yeast strain.Similarly to the selection of antibody clones from the primary antibodylibrary, the AD-scFv containing haploid cells are mated with haploidcells of opposite mating type (e.g. a type) that contains the BD-Targetvector and the reporter gene construct. The parental diploid cells areselected based on expression of the reporter gene and other selectioncriteria as described in detail in Section 2.

[0205] Other PCR-based mutagenesis method can also be used, alone or inconjunction with the poison PCR described above. For example, the PCRamplified V_(H) and V_(L) segments may be digested with DNase to createnicks in the double DNA strand. These nicks can be expanded into gaps byother exonucleases such as Bal 31. The gaps may be then be filled byrandom sequences by using DNA Klenow polymerase at low concentration ofregular substrates dGTP, dATP, dTTP, and dCTP with one substrate (e.g.,dGTP) at a disproportionately high concentration. This fill-in reactionshould produce high frequency mutations in the filled gap regions. Thesemethod of DNase I digestion may be used in conjunction with poison PCRto create highest frequency of mutations in the desired V_(H) and V_(L)segments.

[0206] The PCR amplified V_(H) and V_(L) segments or antibody heavychain and light chain segments may be mutagenized in vitro by using DNAshuffling techniques described by Stemmer (1994) Nature 370:389-391; andStemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. The V_(H),V_(L) or antibody segments from the primary antibody leads are digestedwith DNase I into random fragments which are then reassembled to theiroriginal size by homologous recombination in vitro by using PCR methods.As a result, the diversity of the library of primary antibody leads areincreased as the numbers of cycles of molecular evolution increase invitro.

[0207] The V_(H), V_(L) or antibody segments amplified from the primaryantibody leads may also be mutagenized in vivo by exploiting theinherent ability of mution in pre-B cells. The Ig gene in pre-B cells isspecifically susceptible to a high-rate of mutation in the developmentof pre-B cells. The Ig promoter and enhancer facilitate such high ratemutations in a pre-B cell environment while the pre-B cells proliferate.Accordingly, V_(H) and V_(L) gene segments may be cloned into amammalian expression vector that contains human Ig enhancer andpromoter. This construct may be introduced into a pre-B cell line, suchas 38B9, which allows the mutation of the V_(H) and V_(L) gene segmentsnaturally in the pre-B cells. Liu, X., and Van Ness, B. (1999) Mol.Immunol. 36:461-469. The mutagenized V_(H) and V_(L) segments can beamplified from the cultured pre-B cell line and re-introduced back intothe AD-containing yeast strain via, for example, homologousrecombination.

[0208] The secondary antibody library produced by mutagenesis in vitro(e.g. PCR) or in vivo, i.e., by passing through a mammalian pre-B cellline may be cloned into an expression vector and screened against thesame target protein as in the first round of screening using the primaryantibody library. For example, the expression vectors containing thesecondary antibody library may be transformed into haploid cells of αtype yeast strain. These α cells are mated with haploid cells a typeyeast strain containing the BD-target expression vector and the reportergene construct. The positive interaction of antibodies from thesecondary antibody library is screened by following similar proceduresas described for the selection of the primary antibody leads in yeast.

[0209] 6. Functional Expression and Purification of Selected Antibody

[0210] The humanized antibodies that are generated and selected in thescreening against the target antigen may be functionally expressed inhosts after the V_(H) and V_(L) sequences are operably linked to anexpression control DNA sequence, including naturally-associated orheterologous promoters, in an expression vector. By operably linking thethe V_(H) and V_(L) sequences to an expression control sequence, theV_(H) and V_(L) coding sequences are positioned to ensure thetranscription and translation of these inserted sequences. Theexpression vector may be replicable in the host organism as episomes oras an integral part of the host chromosomal DNA. The expression vectormay also contain selection markers such as antibiotic resistance genes(e.g. neomycin and tetracycline resistance genes) to permit detection ofthose cells transformed with the expression vector.

[0211] Preferably, the expression vector may be a eukaryotic vectorcapable of transforming or transfecting eukaryotic host cells. Once theexpression vector has been incorporated into the appropriate host cells,the host cells are maintained under conditions suitable for high levelexpression of humanized antibody or fragments, such as dcFv, Fab andantibody. The polypeptides expressed are collected and purifieddepending on the expression system used.

[0212] The dcFv, Fab, or fully assembled antibodies selected by usingthe methods of the present invention may be expressed in various scalesin any host system. Examples of host systems include, but are notlimited to, bacteria (e.g. E. coli), yeast (e.g. S. cerevisiae), andmammalian cells (COS). The bacteria expression vector may preferablycontain the bacterial phage T7 promoter and express either the heavychain and/or light chain region of the selected antibody. The yeastexpression vector may contain a constitutive promoter (e.g. ADGIpromoter) or an inducible promoter such as (e.g. GCN4 and Gal 1promoters). All three types of antibody, dcFv, Fab, and full antibody,may be expressed in a yeast expression system.

[0213] The expression vector may be a mammalian express vector that canbe used to express the humanized antibody in mammalian cell culturetransiently or stably. Examples of mammalian cell lines that may besuitable of secreting immunoglobulins include, but are not limited to,various COS cell lines, HeLa cells, myeloma cell lines, CHO cell lines,transformed B-cells and hybridomas.

[0214] Typically, a mammalian expression vector includes certainexpression control sequences, such as an origin of replication, apromoter, an enhancer, as well as necessary processing signals, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Examples of promoters include, butare not limited to, insulin promoter, human cytomegalovirus (CMV)promoter and its early promoter, simian virus SV40 promoter, Roussarcoma virus LTR promoter/enhancer, the chicken cytoplasmic β-actinpromoter, promoters derived from immunoglobulin genes, bovine papillomavirus and adenovirus.

[0215] One or more enhancer sequence may be included in the expressionvector to increase the transcription efficiency. Enhancers arecis-acting sequences of between 10 to 300 bp that increase transcriptionby a promoter. Enhancers can effectively increase transcription whenpositioned either 5′ or 3′ to the transcription unit. They may also beeffective if located within an intron or within the coding sequenceitself. Examples of enhancers include, but are not limited to, SV40enhancers, cytomegalovirus enhancers, polyoma enhancers, the mouseimmunoglobulin heavy chain enhancer and adenovirus enhancers. Themammalian expression vector may also typically include a selectablemarker gene. Examples of suitable markers include, but are not limitedto, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene(TK), or prokaryotic genes conferring antibiotic resistance. The DHFRand TK genes prefer the use of mutant cell lines that lack the abilityto grow without the addition of thymidine to the growth medium.Transformed cells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

[0216] The expression vectors containing the humanized antibodysequences can then be transferred into the host cell by methods known inthe art, depending on the type of host cells. Examples of transfectiontechniques include, but are not limited to, calcium phosphatetransfection, calcium chloride transfection, lipofection,electroporation, and microinjection.

[0217] The humanized antibody sequences may also be inserted into aviral vector such as adenoviral vector that can replicate in its hostcell and produce the antibody in large amounts.

[0218] In particular, the dcFv, Fab, or fully assembled antibody may beexpressed in mammalian cells by using a method described by Persic etal. (1997) Gene, 187:9-18. The mammalian expression vector that isdescribed by Persic and contains EF-α promoter and SV40 replicationorigin is preferably utilized. The SV40 origin allows a high level oftransient expression in cells containing large T antigen such as COScell line. The expression vector may also include secretion signal anddifferent antibiotic markers (e.g. neo and hygro) for integrationselection.

[0219] Once expressed, the humanized antibody may be isolated andpurified by using standard procedures of the art, including ammoniumsulfate precipitation, fraction column chromatography, and gelelectrophoresis. Once purified, partially or to homogeneity as desired,the polypeptides may then be used therapeutically or in developing,performing assay procedures, immunofluorescent staining, and in otherbiomedical and industrial applications. In particular, the antibodiesgenerated by the method of the present invention may be used fordiagnosis and therapy for the treatment of various diseases such ascancer, autoimmune diseases, or viral infections.

[0220] In a preferred embodiment, the humanized antibodies that aregenerated and screened by using the methods of the present invention maybe expressed directly in yeast. According to this embodiment, the heavychain and light chain regions from the selected expression vectors maybe PCR amplified with primers that simultaneously add appropriatehomologous recombination sequences to the PCR products. These PCRsegments of heavy chain and light chain may then be introduced into ayeast strain together with a linearized expression vector containingdesirable promoters, expression tags and other transcriptional ortranslational signals.

[0221] For example, the PCR segments of heavy chain and light chainregions may be homologously recombined with a yeast expression vectorthat already contains a desirable promoter in the upstream and stopcodons and transcription termination signal in the downstream. Thepromoter may be a constitutive expression promoter such as ADH1, or aninducible expression promoter, such as Gal 1, or GCN4 (A. Mimran, I.Marbach, and D. Engelberg, (2000) Biotechniques 28:552-560). The latterinducible promoter may be preferred because the induction can be easilyachieved by adding 3-AT into the medium.

[0222] The yeast expression vector to be used for expression of theantibody may be of any standard strain with nutritional selectionmarkers, such as His 3, Ade 2, Leu 2, Ura 3, Trp 1 and Lys 2. The markerused for the expression of the selected antibody may preferably bedifferent from the AD vector used in the selection of antibody in thetwo-hybrid system. This may help to avoid potential carryover problemassociated with multiple yeast expression vectors.

[0223] For expressing the dcFv antibody in a secreted form in yeast, theexpression vector may include a secretion signal in the 5′ end of theV_(H) and V_(L) segments of the humanized antibody, such as an alphafactor signal and a 5-pho secretion signal. Certain commerciallyavailable vectors that contain a desirable secretion signal may also beused (e.g., pYEX-S1, catalog #6200-1, Clontech, Palo Alto, Calif.).

[0224] The dcFv antibody fragments generated may be analyzed andcharacterized for their affinity and specificity by using methods knownin the art, such as ELISA, western, and immune staining. Those dcFvantibody fragments with reasonably good affinity (with dissociationconstant preferably above 10⁻⁶ M ) and specificity can be used asbuilding blocks in Fab expression vectors, or can be further assembledwith the constant region for full length antibody expression. Thesefully assembled human antibodies may also be expressed in yeast in asecreted form.

[0225] The V_(H) sequence encoding the selected dcFv protein may belinked with the constant regions of a full antibody, C_(H)1, C_(H)2 andC_(H)3. Similarly, the V_(L) sequence may be linked with the constantregion C_(L). The assembly of two units of V_(H)-C_(H)1-C_(H)2-C_(H)3and V_(L)-C_(L) leads to formation of a fully functional antibody.

[0226] The present invention provides a method for producing fullyfunctional humanized antibody in yeast. Fully functional antibodyretaining the rest of the constant regions may have a higher affinity(or avidity) than a dcFv or a Fab. The full antibody should also have ahigher stability, thus allowing more efficient purification of antibodyprotein in large scale.

[0227] The method is provided by exploiting the ability of yeast cellsto uptake and maintain multiple copies of plasmids of the samereplication origin. According to the method, different vectors may beused to express the heavy chain and light chain separately, and yetallows for the assembly of a fully functional antibody in yeast. Thisapproach has been successfully used in a two-hybrid system design wherethe BD and AD vectors are identical in backbone structure except theselection markers are distinct. This approach has been used in atwo-hybrid system design for expressing both BD and AD fusion proteinsin the yeast. The BD and AD vectors are identical in their backbonestructures except the selection markers are distinct. Both vectors canbe maintained in yeast in high copy numbers. Chien, C. T., et al. (1991)“The two-hybrid system: a method to identify and clone genes forproteins that interact with a protein of interest” Proc. Natl. Acad.Sci. USA 88:9578-9582.

[0228] In the present invention, the heavy chain gene and light chaingenes are placed in two different vectors. Under a suitable condition,the V_(H)-C_(H)1-C_(H)2-C_(H)3 and V_(L)-C_(L) sequences are expressedand assembled in yeast, resulting in a fully functional antibody proteinwith two heavy chains and two light chains. This fully functionalantibody may be secreted into the medium and purified directly from thesupernatant.

[0229] The dcFv with a constant region, Fab, or fully assembled antibodycan be purified using methods known in the art. Conventional techniquesinclude, but are not limited to, precipitation with ammonium sulfateand/or caprylic acid, ion exchange chromatography (e.g. DEAE), and gelfiltration chromatography. Delves (1997) “Antibody Production: EssentialTechniques”, New York, John Wiley & Sons, pages 90-113. Affinity-basedapproaches using affinity matrix based on Protein A, Protein G orProtein L may be more efficiency and results in antibody with highpurity. Protein A and protein G are bacterial cell wall proteins thatbind specifically and tightly to a domain of the Fc portion of certainimmunoglobulins with differential binding affinity to differentsubclasses of IgG. For example, Protein G has higher affinities formouse IgG1 and human IgG3 than does Protein A. The affinity of Protein Aof IgG1 can be enhanced by a number of different methods, including theuse of binding buffers with increased pH or salt concentration. ProteinL binds antibodies predominantly through kappa light chain interactionswithout interfering with the antigen-binding site. Chateau et al. (1993)“On the interaction between Protein L and immunoglobulins of variousmammalian species” Scandinavian J. Immunol., 37:399-405. Protein L hasbeen shown to bind strongly to human kappa light chain subclasses I, IIIand IV and to mouse kappa chain subclasses I. Protein L can be used topurify relevant kappa chain-bearing antibodies of all classes (IgG, IgM,IgA, IgD, and IgE) from a wide variety of species, including human,mouse, rat, and rabbit. Protein L can also be used for the affinitypurification of scFv and Fab antibody fragments containing suitablekappa light chains. Protein L-based reagents is commercially availablefrom Actigen, Inc., Cambridge, England. Actigen can provide a line ofrecombinant Protein products, including agarose conjugates for affinitypurification and immobilized forms of recombinant Protein L and A fusionprotein which contains four protein A antibody-binding domains and fourprotein L kappa-binding domains.

[0230] Other affinity matrix may also be used, including those thatexploit peptidomimetic ligands, anti-immunoglobulins, mannan bindingprotein, and the relevant antigen. Peptidomimetic ligands resemblepeptides but they do not correspond to natural peptides. Many ofPeptidomimetic ligands contain unnatural or chemically modified aminoacids. For example, peptidomimetic ligands designed for the affinitypurification of antibodies of the IGA and IgE classes are commerciallyavailable from Tecnogen, Piana di Monte Verna, Italy. Mannan bindingprotein (MBP) is a mannose- and N-acetylglucosamine-specific lectinfound in mammalian sera. This lectin binds IgM. The MBP-agarose supportfor the purification IgM is commercially available from Pierce.

[0231] Immunomagnetic methods that combine an affinity reagent (e.g.protein A or an anti-immunoglobulin) with the ease of separationconferred by paramagnetic beads may be used for purifying the antibodyproduced. Magnetic beads coated with Protein or relevant secondaryantibody may be commercially available from Dynal, Inc., NY; BangsLaboratories, Fishers, Ind.; and Cortex Biochem Inc., San Leandro,Calif.

[0232] Direct expression and purification of the selected antibody inyeast is advantageous in various aspects. As a eukaryotic organism,yeast is more of an ideal system for expressing human proteins thanbacteria or other lower organisms. It is more likely that yeast willmake the dcFv, Fab, or fully assembled antibody in a correctconformation (folded correctly), and will add post-translationmodifications such as correct disulfide bond(s) and glycosylations.

[0233] Yeast has been explored for expressing many human proteins in thepast. Many human proteins have been successfully produced from theyeast, such as human serum albumin (Kang, H. A. et al. (2000) Appl.Microbiol. Biotechnol. 53:578-582) and human telomerase protein and RNAcomplex (Bachand, F., et al. (2000) RNA 6:778-784).

[0234] Yeast has fully characterized secretion pathways. The geneticsand biochemistry of many if not all genes that regulate the pathwayshave been identified. Knowledge of these pathways should aid in thedesign of expression vectors and procedures for isolation andpurification of antibody expressed in the yeast.

[0235] Moreover, yeast has very few secreted proteases. This should keepthe secreted recombinant protein quite stable. In addition, since yeastdoes not secrete many other and/or toxic proteins, the supernatantshould be relatively uncontaminated. Therefore, purification ofrecombinant protein from yeast supernatant should be simple, efficientand economical.

[0236] Additionally, simple and reliable methods have been developed forisolating proteins from yeast cells. Cid, V. J. et al. (1998) “Amutation in the Rho&GAP-encoding gene BEM2 of Saccharomyces cerevisiaeaffects morphogenesis and cell wall functionality” Microbiol. 144:25-36.Although yeast has a relatively thick cell wall that is not present ineither bacterial or mammalian cells, the yeast cells can still keep theyeast strain growing with the yeast cell wall striped from the cells. Bygrowing the yeast strain in yeast cells without the cell wall, secretionand purification of recombinant human antibody may be made more feasibleand efficient.

[0237] By using yeast as host system for expression, a streamlinedprocess can be established to produce recombinant antibodies in fullyassembled and purified form. This may save tremendous time and effortsas compared to using any other systems such as humanization of antibodyin vitro and production of fully human antibody in transgenic animals.

[0238] In summary, the compositions, kits and methods provided by thepresent invention should be very useful for humanized antibodies withhigh affinity and specificity against a wide variety of targetsincluding, but not limited to, soluble proteins (e.g. growth factors,cytokines and chemokines), membrane-bound proteins (e.g. cell surfacereceptors), and viral antigens. The whole process of libraryconstruction, functional screening and expression of highly diverserepertoire of human antibodies can be streamlined, and efficiently andeconomically performed in yeast or displayed on ribosome in a highthroughput and automated manner. The selected proteins can have a widevariety of applications. For example, they can be used in therapeuticsand diagnosis of diseases including, but not limited to, autoimmunediseases, cancer, transplant rejection, infectious diseases andinflammation.

1 9 1 296 DNA Artificial Sequence Consensus human germline VH sequence(DP47) 1 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtccctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggtccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtagcacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaacacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgcgaaaga 296 2 290 DNA Artificial Sequence Consensus human germline VLsequence (DPK22) 2 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgtctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctacttagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatccagcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactctcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggtagctcacctcc 290 3 5 PRT Artificial Sequence Single chain antibody linkersequence G4S 3 Gly Gly Gly Gly Ser 1 5 4 357 DNA Mus musculus 4caggtccagt tgcagcagtc tggagctgag tcggtaaggc ctgggacttc agtgaagata 60tcctgcaagg cttctggcta caccttcact aactactggc taggttgggt aaagcagagg 120cctggacatg gacttgagtg gattggagat atttaccctg gaggtggtta tactaactac 180aatgagaagt tcaaggacaa ggccacactg acaacagaca catcctccag cactgcctac 240atgcagctca gtagcctgac atctgatgac tctgctgtct atttctgtgc aagggactac 300ggtagtaggt actactttga ctactggggc caaggcacca ctctcacagt ctcctca 357 5 119PRT Mus musculus 5 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Ser Val ArgPro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr ThrPhe Thr Asn Tyr 20 25 30 Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His GlyLeu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn TyrAsn Glu Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Thr Ser SerSer Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp SerAla Val Tyr Phe Cys 85 90 95 Ala Arg Asp Tyr Gly Ser Arg Tyr Tyr Phe AspTyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 6 321DNA Mus musculus 6 gatatccaga tgacacagac tacatcctcc ctgtctgcctctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagc aattttttaaactggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctactac acatcaagattacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctcaccattagcaa cctggaacaa 240 gaagatattg ccacttactt ttgccaacag ggtaacacgctgtggacgtt cggtggaggc 300 accaagctgg aaatcaaacg g 321 7 107 PRT Musmusculus 7 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser LeuGly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile SerAsn Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys LeuLeu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn LeuGlu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn ThrLeu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 1058 118 PRT Homo sapiens 8 Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu ValArg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly TyrAla Phe Ser Ser Ser 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly GlnGly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr AsnTyr Asn Gly Lys Phe 50 55 60 Lys Glu Ala Ala Thr Leu Thr Ala Asp Lys SerSer Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Val AspSer Ala Val Tyr Ser Cys 85 90 95 Ala Arg Ser Glu Tyr Trp Gly Asn Tyr TrpAla Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr 115 9 107PRT Homo sapiens 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln SerIle Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro SerArg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln SerTyr Ser Thr Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg100 105

What is claimed is:
 1. A method for screening a library of humanizedantibody in yeast, comprising: expressing a library of humanizedantibodies in yeast cells; expressing a specific target protein in theyeast cells expressing the humanized antibodies; and selecting thoseyeast cells in which a reporter gene is expressed, the expression of thereporter gene being activated by binding of the humanized antibody tothe target protein.
 2. The method of claim 1, wherein a member of thelibrary of humanized antibody contains one or more CDRs of a non-humanantibody grafted into a human antibody framework.
 3. The method of claim1, wherein the library of humanized antibody are generated bymutagenizing a chimeric antibody that contains one or more CDRs of anon-human antibody grafted into a human antibody framework.
 4. A methodfor screening a library of humanized antibody in vitro, comprising:expressing a library of humanized antibodies in vitro; displaying thelibrary of humanized antibodies on ribosome; contacting the ribosomedisplaying the library of humanized antibodies with a target antigen;and selecting those ribosomes that bind to the target antigen,
 5. Themethod of claim 4, wherein the target antigen is immobilized to asubstrate.
 6. The method of claim 4, wherein the target antigen isfluorescence labeled.
 7. A method for screening a library of humanizedantibody in vitro, comprising: transcribing a library of cDNA encoding alibrary of humanized antibodies in vitro to produce a library of mRNA;contacting the library of mRNA with a peptidyl acceptor linker toproduce a library of modified mRNA; translating the library of modifiedmRNA in vitro to produce a library of humanized antibody, wherein thetranslated humanized antibody is covalently linked to the modified mRNAby the peptidyl acceptor linker; and contacting the library of humanizedantibodies linked with the modified mRNA with a target antigen; andselecting the humanized antibody that bind to the target antigen,
 8. Themethod of claim 7, wherein the peptidyl acceptor linker is puromycin.